Rapafucin derivative compounds and methods of use thereof

ABSTRACT

The present disclosure provides macrocyclic compounds inspired by the immunophilin ligand family of natural products FK506 and rapamycin. The generation of a Rapafucin library of macrocyles that contain FK506 and rapamycin binding domains should have great potential as new leads for developing drugs to be used for treating diseases.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S. patent application Ser. No. 16/074,017, filed on Jul. 30, 2018, which is a US National Phase Application of International Application No. PCT/US2017/016481, filed on Feb. 3, 2017, which claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/291,437, filed on Feb. 4, 2016. The entire content of all three of which are incorporated herein by reference.

STATEMENT OF GOVERNMENT SUPPORT

This disclosure was made with government support under National Institutes of Health grant DP1CA174428. The government has certain rights in this disclosure.

BACKGROUND INFORMATION

The macrocyclic natural products FK506 and rapamycin are approved immunosuppressive drugs with important biological activities. Both have been shown to inhibit T cell activation, albeit with distinct mechanisms. In addition, rapamycin has been shown to have strong anti-proliferative activity. FK506 and rapamycin share an extraordinary mode of action; they act by recruiting an abundant and ubiquitously expressed cellular protein, the prolyl cis-trans isomerase FKBP, and the binary complexes subsequently bind to and allosterically inhibit their target proteins calcineurin and mTOR, respectively. Structurally, FK506 and rapamycin share a similar FKBP-binding domain but differ in their effector domains. In FK506 and rapamycin, nature has taught us that switching the effector domain of FK506 to that in rapamycin, it is possible to change the targets from calcineurin to mTOR. The generation of a Rapafucin library of macrocyles that contain FK506 and rapamycin binding domains should have great potential as new leads for developing drugs to be used for treating diseases.

With the completion of the sequencing and annotation of the human genome, a complete catalog of all human proteins encoded in the genome is now available. The functions of a majority of these proteins, however, remain unknown. One way to elucidate the functions of these proteins is to find small molecule ligands that specifically bind to the proteins of interest and perturb their biochemical and cellular functions. Thus, a major challenge for chemical biologists today is to discover new small molecule probes for new proteins to facilitate the elucidation of their functions. The recent advance in the development of protein chips has offered an exciting new opportunity to simultaneously screen chemical libraries against nearly the entire human proteome. A single chip, in the form of a glass slide, is sufficient to display an entire proteome in duplicate arrays. Recently, a protein chip with 17,000 human proteins displayed on a single slide has been produced. A major advantage of using human protein chips for screening is that the entire displayed proteome can be interrogated at once in a small volume of assay buffer (<3 mL). Screening of human protein chips, however, is not yet feasible with most, if not all, existing chemical libraries due to the lack of a universal readout for detecting the binding of a ligand to a protein on these chips. While it is possible to add artificial tags to individual compounds in a synthetic library, often the added tags themselves interfere with the activity of ligands. Thus, there remains a need for new compounds and methods for screening chemical libraries against the human proteome.

SUMMARY

The present disclosure is directed to a library of Rapafucin compounds, methods of making these compounds, and methods of using the same. The present disclosure is further directed to DNA-encoded libraries of hybrid cyclic molecules, and more specifically to DNA-encoded libraries of hybrid cyclic compounds based on the immunophilin ligand family of natural products FK506 and rapamycyin.

In some embodiments, the Rapafucin compounds in the present disclosure can have a structure according to Formula (V) or an optically pure stereoisomer or pharmaceutically acceptable salt thereof.

Wherein L is selected from the groups in Table 1; A is CH₂, NH, NMe, O, S(O)₂ or S; each D is independently O, NMe, or NH; E is CH or N.

Each of R¹, R², R³, and R⁴ can be independently selected from the group consisting of H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, CO₂C₁₋₂₀alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₁₋₁₀alkoxy, C₆₋₁₅aryl, C₆₋₁₅aryloxy, C₆₋₁₅arylthio, C₂₋₁₀carboxyl, C₁₋₁₀alkylamino, thiol, C₁₋₁₀alkylthio, C₁₋₁₀alkyldisulfide, C₆₋₁₅arylthio, C₁₋₁₀heteroarylthio, (C₃₋₈cycloalkyl)thio, C₂₋₁₀heterocyclylthio, sulfonyl, C₁₋₁₀alkylsulfonyl, amido, C₁₋₁₀alkylamido, selenol, C₁₋₁₀alkylselenol, C₆₋₁₅arylselenol, C₁₋₁₀heteroarylselenol, (C₃₋₈cycloalkyl)selenol, C₂₋₁₀heterocyclylselenol, guanidino, C₁₋₁₀alkylguanidino, urea, C₁₋₁₀alkylurea, ammonium, C₁₋₁₀alkylammonium, cyano, C₁₋₁₀alkylcyano, C₁₋₁₀alkylnitro, adamantine, phosphonate, C₁₋₁₀alkylphosphonate, and C₆₋₁₅arylphosphonate, each of the above can be optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₂₀alkyl, substituted C₂₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

n is an integer selected from 0 to 4; m is an integer selected from 0 to 5; each p is independently an integer selected from 0 to 2; q is an integer selected from 1 to 10.

Or any R⁴ forms a cyclic structure formed with any R³, the cyclic structure so formed is selected from the group consisting of C₂₋₁₀heterocyclyl and C₁₋₁₀heteroaryloptionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

In some embodiments, q can be 1. In some embodiments, q can be 2. In some embodiments, q can be 3. In some embodiments, q can be 4. In some embodiments, q can be 5. In some embodiments, q can be 6. In some embodiments, q can be 7. In some embodiments, q can be 8. In some embodiments, q can be 9. In some embodiments, q can be 10. In specific embodiments, q is 3 or 4.

Further provided herein is a macrocyclic compound of Formula (XII) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof:

In some embodiments, Ring A is a 5-10 membered aryl, cycloalkyl, heteroaryl or heterocycloalkyl, optionally substituted with 1-17 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin; J is independently at each occurrence selected from the group consisting of —C(O)NR⁶—.

wherein R⁶ is each hydrogen, alkyl, arylalkyl,

wherein R is aryl, alkyl, or arylalkyl; R′ is hydrogen, alkyl, arylalkyl, or haloalkyl; D is independently at each occurrence an oligonucleotide; L^(b) and L^(c) are independently at each occurrence selected from the group consisting of bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)OC(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)—S—C₁₋₆ alkylene (CH₂)_(n)—, and optionally substituted (CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or 2 groups independently selected from the group consisting of of halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl, arylalkyl, R^(N) or and

wherein R^(N) is aryl, alkyl, or arylalkyl; X is O, S or NR⁸, wherein R⁸ is hydrogen, hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ are each independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionally substituted with 1-10 substituents, each of which is selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino, arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen, hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is each independently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, arylalkyl, or heteroaryl; Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸, CR¹⁸, N, or and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl;

L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond, —O—, —NR¹⁹—, —SO—, —SO₂—, (CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6 membered heteroaryl, optionally substituted with 1-4 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxyl, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino and

wherein each R¹⁹, R²⁰, and R²¹ is independently is selected from the group consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴ are each independently hydrogen or alkyl;

n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula (XIIa):

In some embodiments, each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i) is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ is independently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ is hydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸ are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is independently selected from the group consisting of hydrogen, halo, amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnatural amino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

or wherein the Effector Domain has Formula (XIIc):

wherein each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R²⁹ is a hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³² are each independently hydrogen or alkyl; X³ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIId):

wherein X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIe):

wherein R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; provided that when R is

L is ethylene, X is O, W is

V is

Z is

-L⁶-L⁷-L⁸-is

then -L¹-L²-L³-L⁴-L⁵-is not

and; wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L L⁷ and L⁸ is Ring C substituted with at least one

or wherein at least one of the linking groups selected from Table 1 is substituted with at least one

In another aspect, provided herein is a tagged macrocyclic compound of a compound of Formula (XII):

with a compound of Formula (XIV):

Q′-L^(c)-D   Formula (XIV)

Ring A is a 5-10 membered aryl, cycloalkyl, heteroaryl or heterocycloalkyl, optionally substituted with 1-17 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin;

L^(b) and L^(c) are independently selected from the group consisting of a bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)OC(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)—S—C₁₋₆ alkylene-(CH₂)_(n)—, and optionally substituted (CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or 2 groups independently selected from the group consisting of of halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl;

Q and Q′ are independently selected from the group consisting of —N₃, —C≡CH, NR⁶R⁷, —COOH, —ONH₂, —SH, —NH₂,

—(C═O)R′,

wherein R⁶ and R⁷ is each independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; and R′ is hydrogen, alkyl, arylalkyl, or haloalkyl; X is O, S or NR⁸, wherein R⁸ is hydrogen, hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ are each independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionally substituted with 1-10 substituents, each of which is selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino, arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen, hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is each independently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, arylalkyl, or heteroaryl;

Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸, CR¹⁸, N, and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl;

L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond, —O—, —NR¹⁹—, —SO—, —SO₂—, —(CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6 membered heteroaryl, optionally substituted with 1-4 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino and

wherein R¹⁹ is selected from the group consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴ are each independently hydrogen or alkyl;

n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula (XIIa):

each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i) is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ is independently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ is hydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸ are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is independently selected from the group consisting of hydrogen, halo, amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnatural amino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

or wherein the Effector Domain has Formula (XIIc):

each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R²⁹ is hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³² are each independently hydrogen or alkyl; X³ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIId):

X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIe):

R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X¹⁸ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; and provided that when Ring A is

L^(a) is ethylene, X is O, W is

V is

V² is

Z is

-L⁶-L⁷-L⁸ is

and -L¹-L²-L³-L⁴-L⁵-is not

D is an oligonucleotide; wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring C substituted with at least one

or wherein at least one of the linking groups selected from Table 1 is substituted with at least one

DETAILED DESCRIPTION

Nature is a bountiful source of bioactive small molecules that display a dizzying array of cellular activities thanks to the evolution process over billions of years. Rapamycin and FK506 comprise a unique structural family of macrocyclic natural products with an extraordinary mode of action. On entering cells, both compounds form binary complexes with FKBP12 as well as other members of the FKBP family. The FKBP12-rapamycin complex can then bind to mTOR and block its kinase activity towards downstream substrates such as p70S6K and 4E-BP, while the FKBP12-FK506 complex interacts with calcineurin, a protein phosphatase whose inhibition prevents calcium-dependent signaling and T cell activation. The ability of rapamycin and FK506 to bind FKBPs confers a number of advantages for their use as small molecule probes in biology as well as drugs in medicine. First, the binding of both rapamycin and FK506 to FKBP dramatically increases their effective sizes, allowing for allosteric blockade of substrates to the active sites of mTOR or calcineurin through indirect disruption of protein-protein interactions. Second, the abundance and ubiquitous expression of intracellular FKBPs serves to enrich rapamycin and FK506 in the intracellular compartment and maintain their stability. Third, as macrocycles, FK506 and rapamycin are capable of more extensive interactions with proteins than smaller molecules independent of their ability to bind FKBP. Last, but not least, the high-level expression of FKBPs in blood cells renders them reservoirs and carriers of the drugs for efficient delivery in vivo. It is thus not surprising that both rapamycin and FK506 became widely used drugs in their natural forms without further chemical modifications.

Both rapamycin and FK506 can be divided into two structural and functional domains: an FKBP-binding domain (FKBD) and an effector domain that mediates interaction with mTOR or calcineurin, respectively. The structures of the FKBDs of rapamycin and FK506 are quite similar, but their effector domains are different, accounting for their exclusive target specificity. The presence of the separable and modular structural domains of FK506 and rapamycin have been extensively exploited to generate new analogues of both FK506 and rapamycin, including chemical inducers of dimerization and a large number of rapamycin analogues, known as rapalogs, to alter the specificity of rapamycin for the mutated FKBP-rapamycin binding domain of mTOR and to improve the toxicity and solubility profiles of rapamycin. The existence of two distinct FKBD containing macrocycles with distinct target specificity also raised the intriguing question of whether replacing the effector domains of rapamycin or FK506 could further expand the target repertoire of the resultant macrocycles. In their pioneering work, Chakraborty and colleagues synthesized several rapamycin-peptide hybrid molecules, which retained high affinity for FKBP but showed no biological activity. More recently, we and others independently attempted to explore this possibility by making larger libraries of the FKBD-containing macrocycles. In one study, a much larger library of FKBD-containing macrocycles was made with a synthetic mimic of FKBD, but the resultant macrocycles suffered from a significant loss in binding affinity for FKBP12, probably accounting for the lack of bioactive compounds from that library. Using a natural FKBD extracted from rapamycin, we also observed a significant loss in FKBP binding affinity on formation of macrocycles (vide infra).

A Rapafucin library was synthesized as described in WO2017/136708. Rapadocin compound and analogs thereof are disclosed in WO2017/136717, which are used for inhibiting human equilibrative nucleoside transporter 1 (ENTI). Rapaglutins and analogs thereof are disclosed in WO2017/136731, which are used as inhibitors of cell proliferation and useful for the treatment of cancer. Approximately 45,000 compounds were generated and ongoing screening of the library as described in WO2018/045250 identified several compounds as being inhibitors of MIF nuclease activity. All of these references are incorporated herein by reference.

In a continuing effort to explore the possibility to using FKBD containing macrocycles to target new proteins, we attempted to optimize and succeeded in identifying FKBDs that allowed for significant retention of binding affinity for FKBP12 upon incorporation into macrocycles. We also established a facile synthetic route for parallel synthesis of a large number of FKBD-containing macrocycles.

Below are some acronyms used in the present disclosure. 2-MeTHF refers to 2-methyltetrahydrofuran; DMF refers to dimethylformamide; DMSO refers to dimethyl sulfoxide; DCM refers to dichloromethane; HATU refers to 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; DIEA refers to N, N-Diisopropylethylamine; TFA refers to trifluoroacetic acid; Fmoc refers to fluorenylmethyloxycarbonyl; MeOH refers to methanol; EtOAc refers to ethyl acetate; MgSO4 refers to magnesium sulfate; COMU-PF6 refers to (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate; CAN refers to acetonitrile; Oxyma refers to ethyl cyanohydroxyiminoacetate; LC-MS refers to liquid chromatography-mass spectrometry; T3P refers to n-propanephosphonic acid anhydride; SPPS refers to solid-phase peptide synthesis.

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements. The term “about” will be understood by persons of ordinary skill in the art. Whether the term “about” is used explicitly or not, every quantity given herein refers to the actual given value, and it is also meant to refer to the approximation to such given value that would be reasonably inferred based on the ordinary skill in the art.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment. A person of ordinary skill in the art would recognize that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, pentavalent carbon, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All sequences provided in the disclosed Genbank Accession numbers are incorporated herein by reference. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Alkyl groups refer to univalent groups derived from alkanes by removal of a hydrogen atom from any carbon atom, which include straight chain and branched chain with from 1 to 12 carbon atoms, and typically from 1 to about 10 carbons or in some embodiments, from 1 to about 6 carbon atoms, or in other embodiments having 1, 2, 3 or 4 carbon atoms. Examples of straight chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl groups. Examples of branched chain alkyl groups include, but are not limited to isopropyl, isobutyl, sec-butyl and tert-butyl groups. Alkyl groups may be substituted or unsubstituted. Representative substituted alkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted. As used herein, the term alkyl, unless otherwise stated, refers to both cyclic and noncyclic groups.

The terms “cyclic alkyl” or “cycloalkyl” refer to univalent groups derived from cycloalkanes by removal of a hydrogen atom from a ring carbon atom. Cycloalkyl groups are saturated or partially saturated non-aromatic structures with a single ring or multiple rings including isolated, fused, bridged, and spiro ring systems, having 3 to 14 carbon atoms, or in some embodiments, from 3 to 12, or 3 to 10, or 3 to 8, or 3, 4, 5, 6 or 7 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted. Examples of monocyclic cycloalkyl groups include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups. Examples of multi-cyclic ring systems include, but are not limited to, bicycle[4.4.0]decane, bicycle[2.2.1]heptane, spiro[2.2]pentane, and the like. (Cycloalkyl)oxy refers to —O— cycloalkyl. (Cycloalkyl)thio refers to —S-cycloalkyl. This term also encompasses oxidized forms of sulfur, such as —S(O)-cycloalkyl, or —S(O)₂-cycloalkyl.

Alkenyl groups refer to straight and branched chain and cycloalkenyl groups as defined above, with one or more double bonds between two carbon atoms. Alkenyl groups may have 2 to about 12 carbon atoms, or in some embodiment from 1 to about 10 carbons or in other embodiments, from 1 to about 6 carbon atoms, or 1, 2, 3 or 4 carbon atoms in other embodiments. Alkenyl groups may be substituted or unsubstituted. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted. Examples of alkenyl groups include, but are not limited to, vinyl, allyl, —CH═CH(CH₃), —CH═C(CH₃)₂, —C(CH₃)═CH₂, cyclopentenyl, cyclohexenyl, butadienyl, pentadienyl, and hexadienyl, among others.

Alkynyl groups refer to straight and branched chain and cycloalknyl groups as defined above, with one or more triple bonds between two carbon atoms. Alkynyl groups may have 2 to about 12 carbon atoms, or in some embodiment from 1 to about 10 carbons or in other embodiments, from 1 to about 6 carbon atoms, or 1, 2, 3 or 4 carbon atoms in other embodiments. Alkynyl groups may be substituted or unsubstituted. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted. Exemplary alkynyl groups include, but are not limited to, ethynyl, propargyl, and —C≡C(CH₃), among others.

Aryl groups are cyclic aromatic hydrocarbons that include single and multiple ring compounds, including multiple ring compounds that contain separate and/or fused aryl groups. Aryl groups may contain from 6 to about 18 ring carbons, or in some embodiments from 6 to 14 ring carbons or even 6 to 10 ring carbons in other embodiments. Aryl group also includes heteroaryl groups, which are aromatic ring compounds containing 5 or more ring members, one or more ring carbon atoms of which are replaced with heteroatom such as, but not limited to, N, O, and S. Aryl groups may be substituted or unsubstituted. Representative substituted aryl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted. Aryl groups include, but are not limited to, phenyl, biphenylenyl, triphenylenyl, naphthyl, anthryl, and pyrenyl groups. Aryloxy refers to —O-aryl. Arylthio refers to —S-aryl, wherein aryl is as defined herein. This term also encompasses oxidized forms of sulfur, such as —S(O)-aryl, or —S(O)₂-aryl. Heteroaryloxy refers to —O-heteroaryl. Heteroarylthio refers to —S-heteroaryl. This term also encompasses oxidized forms of sulfur, such as —S(O)-heteroaryl, or —S(O)₂-heteroaryl.

Suitable heterocyclyl groups include cyclic groups with atoms of at least two different elements as members of its rings, of which one or more is a heteroatom such as, but not limited to, N, O, or S. Heterocyclyl groups may include 3 to about 20 ring members, or 3 to 18 in some embodiments, or about 3 to 15, 3 to 12, 3 to 10, or 3 to 6 ring members. The ring systems in heterocyclyl groups may be unsaturated, partially saturated, and/or saturated. Heterocyclyl groups may be substituted or unsubstituted. Representative substituted heterocyclyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di-, or tri-substituted. Exemplary heterocyclyl groups include, but are not limited to, pyrrolidinyl, tetrahydrofuryl, dihydrofuryl, tetrahydrothienyl, tetrahydrothiopyranyl, piperidyl, morpholinyl, thiomorpholinyl, thioxanyl, piperazinyl, azetidinyl, aziridinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, imidazolyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, oxetanyl, thietanyl, homopiperidyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxolanyl, dioxanyl, purinyl, quinolizinyl, cinnolinyl, phthalazinyl, pteridinyl, and benzothiazolyl groups. Heterocyclyloxy refers to —O-heterocycyl. Heterocyclylthio refers to —S-heterocycyl. This term also encompasses oxidized forms of sulfur, such as —S(O)-heterocyclyl, or —S(O)₂-heterocyclyl.

Polycyclic or polycyclyl groups refer to two or more rings in which two or more carbons are common to the two adjoining rings, wherein the rings are “fused rings”; if the rings are joined by one common carbon atom, these are “spiro” ring systems. Rings that are joined through non-adjacent atoms are “bridged” rings. Polycyclic groups may be substituted or unsubstituted. Representative polycyclic groups may be substituted one or more times.

Halogen groups include F, Cl, Br, and I; nitro group refers to —NO₂; cyano group refers to —CN; isocyano group refers to —N—C; epoxy groups encompass structures in which an oxygen atom is directly attached to two adjacent or non-adjacent carbon atoms of a carbon chain or ring system, which is essentially a cyclic ether structure. An epoxide is a cyclic ether with a three-atom ring.

An alkoxy group is a substituted or unsubstituted alkyl group, as defined above, singular bonded to oxygen. Alkoxy groups may be substituted or unsubstituted. Representative substituted alkoxy groups may be substituted one or more times. Exemplary alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, isopropoxy, sec-butoxy, tert-butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, and cyclohexyloxy groups.

Thiol refers to —SH. Thiocarbonyl refers to (═S). Sulfonyl refers to —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl, —SO₂— substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl, —SO₂-heterocyclyl, and —SO₂— substituted heterocyclyl. Sulfonylamino refers to —NR^(a)SO₂alkyl, —NR^(a)SO₂-substituted alkyl, —NR^(a)SO₂cycloalkyl, —NR^(a)SO₂substituted cycloalkyl, —NR^(a)SO₂aryl, —NR^(a)SO₂substituted aryl, —NR^(a)SO₂heteroaryl, —NR^(a)SO₂ substituted heteroaryl, —NR^(a)SO₂heterocyclyl, —NR^(a)SO₂ substituted heterocyclyl, wherein each R^(a) independently is as defined herein.

Carboxyl refers to —COOH or salts thereof. Carboxyester refers to —C(O)O-alkyl, —C(O)O— substituted alkyl, —C(O)O-aryl, —C(O)O-substituted aryl, —C(O)β-cycloalkyl, —C(O)O— substituted cycloalkyl, —C(O)O-heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O— heterocyclyl, and —C(O)O-substituted heterocyclyl. (Carboxyester)amino refers to —NR^(a)—C(O)O-alkyl, —NR^(a)—C(O)O-substituted alkyl, —NR^(a)—C(O)O-aryl, —NR^(a)—C(O)O-substituted aryl, —NR^(a)—C(O)₃-cycloalkyl, —NR^(a)—C(O)O-substituted cycloalkyl, —NR^(a)—C(O)O-heteroaryl, —NR^(a)—C(O)O-substituted heteroaryl, —NR^(a)—C(O)O-heterocyclyl, and —NR^(a)—C(O)O-substituted heterocyclyl, wherein R^(a) is as recited herein. (Carboxyester)oxy refers to —O—C(O)O-alkyl, —O—C(O)O— substituted alkyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)₃-cycloalkyl, —O—C(O)O-substituted cycloalkyl, —O—C(O)O-heteroaryl, —O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclyl, and —O—C(O)O-substituted heterocyclyl. Oxo refers to (═O).

The terms “amine” and “amino” refer to derivatives of ammonia, wherein one of more hydrogen atoms have been replaced by a substituent which include, but are not limited to alkyl, alkenyl, aryl, and heterocyclyl groups. Carbamate groups refers to —O(C═O)NR₁R₂, where R₁ and R₂ are independently hydrogen, aliphatic groups, aryl groups, or heterocyclyl groups.

Aminocarbonyl refers to —C(O)N(R^(b))₂, wherein each R^(b) independently is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also, each R^(b) may optionally be joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, provided that both R^(b) are not both hydrogen. Aminocarbonylalkyl refers to -alkylC(O)N(R^(b))₂, wherein each R^(b) independently is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also, each R^(b) may optionally be joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, provided that both R^(b) are not both hydrogen. Aminocarbonylamino refers to —NR^(a)C(O)N(R^(b))₂, wherein R^(a) and each R^(b) are as defined herein. Aminodicarbonylamino refers to —NR^(a)C(O)C(O)N(R^(b))₂, wherein R^(a) and each R^(b) are as defined herein. Aminocarbonyloxy refers to —O—C(O)N(R^(b))₂, wherein each R^(b) independently is as defined herein. Aminosulfonyl refers to —SO₂N(R^(b))₂, wherein each R^(b) independently is as defined herein.

Imino refers to —N═R^(c) wherein R^(c) may be selected from hydrogen, aminocarbonylalkyloxy, substituted aminocarbonylalkyloxy, aminocarbonylalkylamino, and substituted aminocarbonylalkylamino.

The term “optionally substituted” means the anteceding group may be substituted or unsubstituted. When substituted, the substituents of an “optionally substituted” group may include, without limitation, one or more substituents independently selected from the following groups or a particular designated set of groups, alone or in combination: lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, lower acyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester, lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, lower alkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lower haloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonic acid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H, pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Two substituents may be joined together to form a fused five-, six-, or seven-membered carbocyclic or heterocyclic ring consisting of zero to three heteroatoms, for example forming methylenedioxy or ethylenedioxy. An optionally substituted group may be unsubstituted (e.g., —CH₂CH₃), fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F) or substituted at a level anywhere in-between fully substituted and monosubstituted (e.g., —CH₂CF₃). Where substituents are recited without qualification as to substitution, both substituted and unsubstituted forms are encompassed. Where a substituent is qualified as “substituted,” the substituted form is specifically intended. Additionally, different sets of optional substituents to a particular moiety may be defined as needed; in these cases, the optional substitution will be as defined, often immediately following the phrase, “optionally substituted with.”

Pharmaceutically acceptable salts of compounds described herein include conventional nontoxic salts or quaternary ammonium salts of a compound, e.g., from non-toxic organic or inorganic acids. For example, such conventional nontoxic salts include those derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and the like. In other cases, described compounds may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. These salts can likewise be prepared in situ in the administration vehicle or the dosage form manufacturing process, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

The term “treatment” is used interchangeably herein with the term “therapeutic method” and refers to both 1) therapeutic treatments or measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic conditions, disease or disorder, and 2) and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disease or disorder as well as those who may ultimately acquire the disorder (i.e., those needing preventive measures).

The term “subject” as used herein refers to any individual or patient to which the subject methods are performed. Generally, the subject is human, although as will be appreciated by those in the art, the subject may be an animal.

The terms “therapeutically effective amount”, “effective dose”, “therapeutically effective dose”, “effective amount,” or the like refer to the amount of a subject compound that will elicit the biological or medical response in a tissue, system, animal or human that is being sought by administering said compound. Generally, the response is either amelioration of symptoms in a patient or a desired biological outcome. Such amount should be sufficient to inhibit MIF activity.

Also disclosed herein are pharmaceutical compositions including compounds with the structures of Formula (I). The term “pharmaceutically acceptable carrier” refers to a non-toxic carrier that may be administered to a patient, together with a compound of this disclosure, and which does not destroy the pharmacological activity thereof. Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this disclosure include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat and self-emulsifying drug delivery systems (SEDDS) such as α-tocopherol, polyethyleneglycol 1000 succinate, or other similar polymeric delivery matrices.

In pharmaceutical composition comprising only the compounds described herein as the active component, methods for administering these compositions may additionally comprise the step of administering to the subject an additional agent or therapy. Such therapies include, but are not limited to, an anemia therapy, a diabetes therapy, a hypertension therapy, a cholesterol therapy, neuropharmacologic drugs, drugs modulating cardiovascular function, drugs modulating inflammation, immune function, production of blood cells; hormones and antagonists, drugs affecting gastrointestinal function, chemotherapeutics of microbial diseases, and/or chemotherapeutics of neoplastic disease. Other pharmacological therapies can include any other drug or biologic found in any drug class. For example, other drug classes can comprise allergy/cold/ENT therapies, analgesics, anesthetics, anti-inflammatories, antimicrobials, antivirals, asthma/pulmonary therapies, cardiovascular therapies, dermatology therapies, endocrine/metabolic therapies, gastrointestinal therapies, cancer therapies, immunology therapies, neurologic therapies, ophthalmic therapies, psychiatric therapies or rheumatologic therapies. Other examples of agents or therapies that can be administered with the compounds described herein include a matrix metalloprotease inhibitor, a lipoxygenase inhibitor, a cytokine antagonist, an immunosuppressant, a cytokine, a growth factor, an immunomodulator, a prostaglandin or an anti-vascular hyperproliferation compound.

The term “therapeutically effective amount” as used herein refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) Preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, (2) Inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and (3) Ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).

As used herein, the terms “combination,” “combined,” and related terms refer to the simultaneous or sequential administration of therapeutic agents in accordance with this disclosure. For example, a described compound may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form. Accordingly, the present disclosure provides a single unit dosage form comprising a described compound, an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle. Two or more agents are typically considered to be administered “in combination” when a patient or individual is simultaneously exposed to both agents. In many embodiments, two or more agents are considered to be administered “in combination” when a patient or individual simultaneously shows therapeutically relevant levels of the agents in a particular target tissue or sample (e.g., in brain, in serum, etc.).

When the compounds of this disclosure are administered in combination therapies with other agents, they may be administered sequentially or concurrently to the patient. Alternatively, pharmaceutical or prophylactic compositions according to this disclosure comprise a combination of ivermectin, or any other compound described herein, and another therapeutic or prophylactic agent. Additional therapeutic agents that are normally administered to treat a particular disease or condition may be referred to as “agents appropriate for the disease, or condition, being treated.”

The compounds utilized in the compositions and methods of this disclosure may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those, which increase biological penetration into a given biological system (e.g., blood, lymphatic system, or central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and/or alter rate of excretion.

According to a preferred embodiment, the compositions of this disclosure are formulated for pharmaceutical administration to a subject or patient, e.g., a mammal, preferably a human being. Such pharmaceutical compositions are used to ameliorate, treat or prevent any of the diseases described herein in a subject.

Agents of the disclosure are often administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. See Remington's Pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa., 1980). The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

In some embodiments, the present disclosure provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of a described compound, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents for use in treating the diseases described herein, including, but not limited to stroke, ischemia, Alzheimer's, ankylosing spondylitis, arthritis, osteoarthritis, rheumatoid arthritis, psoriatic arthritis, asthma atherosclerosis, Crohn's disease, colitis, dermatitis diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome, systemic lupus erythematous, nephritis, ulcerative colitis and Parkinson's disease. While it is possible for a described compound to be administered alone, it is preferable to administer a described compound as a pharmaceutical formulation (composition) as described herein. Described compounds may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

As described in detail, pharmaceutical compositions of the present disclosure may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream or foam; sublingually; ocularly; transdermally; or nasally, pulmonary and to other mucosal surfaces.

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations for use in accordance with the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient, which can be combined with a carrier material, to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound, which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of active ingredient. In some embodiments, this amount will range from about 5% to about 70%, from about 10% to about 50%, or from about 20% to about 40%.

In certain embodiments, a formulation as described herein comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present disclosure. In certain embodiments, an aforementioned formulation renders orally bioavailable a described compound of the present disclosure.

Methods of preparing formulations or compositions comprising described compounds include a step of bringing into association a compound of the present disclosure with the carrier and, optionally, one or more accessory ingredients. In general, formulations may be prepared by uniformly and intimately bringing into association a compound of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80, Cremophor RH40, and Cremophor E1) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as those described in Pharmacopeia Helvetica, or a similar alcohol. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.

In some cases, in order to prolong the effect of a drug, it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices of the described compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

The pharmaceutical compositions of this disclosure may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers, which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions and solutions and propylene glycol are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

Formulations described herein suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present disclosure as an active ingredient. Compounds described herein may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), an active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants; absorbents, such as kaolin and bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made in a suitable machine in which a mixture of the powdered compound is moistened with an inert liquid diluent. If a solid carrier is used, the preparation can be in tablet form, placed in a hard gelatin capsule in powder or pellet form, or in the form of a troche or lozenge. The amount of solid carrier will vary, e.g., from about 25 to 800 mg, preferably about 25 mg to 400 mg. When a liquid carrier is used, the preparation can be, e.g., in the form of a syrup, emulsion, soft gelatin capsule, sterile injectable liquid such as an ampule or nonaqueous liquid suspension. Where the composition is in the form of a capsule, any routine encapsulation is suitable, for example, using the aforementioned carriers in a hard gelatin capsule shell.

Tablets and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may alternatively or additionally be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration of compounds of the disclosure include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

The pharmaceutical compositions of this disclosure may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this disclosure with a suitable non-irritating excipient, which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of this disclosure is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this disclosure include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this disclosure may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-administered transdermal patches are also included in this disclosure.

The pharmaceutical compositions of this disclosure may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.

Transdermal patches have the added advantage of providing controlled delivery of a compound of the present disclosure to the body. Dissolving or dispersing the compound in the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of the compound across the skin. Either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel can control the rate of such flux.

Examples of suitable aqueous and nonaqueous carriers, which may be employed in the pharmaceutical compositions of the disclosure, include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

Such compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Inclusion of one or more antibacterial and/orantifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like, may be desirable in certain embodiments. It may alternatively or additionally be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents, which delay absorption such as aluminum monostearate and gelatin.

In certain embodiments, a described compound or pharmaceutical preparation is administered orally. In other embodiments, a described compound or pharmaceutical preparation is administered intravenously. Alternative routes of administration include sublingual, intramuscular, and transdermal administrations.

When compounds described herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Preparations described herein may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for the relevant administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.

Such compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, compounds described herein which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

The terms “administration of” and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization.

The crystal structures of the FKBP-FK506-calcineurin and FKBP-rapamycin-TOR complexes revealed that both FK506 and rapamycin can be divided into two functional domains, the “FKBP-binding domain” (FKBD) and the “effector” domain, which mediate their interactions with calcineurin and TOR, respectively. While there are extensive protein-protein interactions between FKBP and calcinerin in their ternary complex, there are far fewer interactions between FKBP and TOR, suggesting that the key role of FKBP in the inhibition of TOR by rapamycin is to bind to FKBD of the drug and present its effector domain to TOR.

A comparison of the structures of FK506 and rapamycin reveal that they share a nearly identical FKBD but each possesses a distinct effector domain. By swapping the effector domain of FK506 with that of rapamycin, it is possible to change the target from calcineurin to TOR, which bears no sequence, functional or structural similarities to each other. In addition, other proteins may be targeted by grafting new structures onto the FKBD of FK506 and rapamycin. Thus, the generation of new compounds with new target specificity may be achieved by grafting a sufficiently large combinatorial library onto FKBD in conjunction with proteome-wide screens through which each compound in the library is tested against every protein in the human proteome.

In some embodiments, provided herein is a macrocyclic compound according to Formula (I), which includes an FKBD, an effector domain, a first linker, and a second linker, wherein the FKBD, the effector domain, the first linker, and the second linker together form a macrocycle.

In some embodiments, provided herein is a macrocyclic compound according to Formula (II) or an optically pure stereoisomer or pharmaceutically acceptable salt thereof.

B can be CH₂, NH, NMe, O, S, or S(O)₂; X can be O, NH or NMe; E can be CH or N; n is an integer selected from 0 to 4; m is an integer selected from 1 to 10. AA in this formula represents natural and unnatural amino acids, each of which can be selected from Table 4 below.

In some embodiments, m can be 1. In some embodiments, m can be 2. In some embodiments, m can be 3. In some embodiments, m can be 4. In some embodiments, m can be 5. In some embodiments, m can be 6. In some embodiments, m can be 7. In some embodiments, m can be 8. In some embodiments, m can be 9. In some embodiments, m can be 10. In specific embodiment, m is 3 or 4.

Each R¹ is selected from the group consisting of H, halogen, hydroxyl, C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, and CO₂C₁₋₂₀alkyl. R² is selected from the group consisting of C₆₋₁₅aryl and C₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

V is

Z is a bond,

wherein R³ and R⁴ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR⁵, CR⁵, N, and NR⁵, wherein R⁵ is hydrogen or alkyl.

Each of L¹, L², or L³ can be selected from the group consisting of the structures shown in Table 1 below.

TABLE 1 The linker structures. optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C₁₋₆ alkylene —(CH₂)_(n)C₂₋₆ alkenylene —(CH₂)_(n)C₃₋₆ cycloalkylene —(CH₂)_(n)C₃₋₆ cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC₁₋₆ alkylene —(CH₂)_(n)OC₂₋₆ —(CH₂)_(n)OC₃₋₆ cycloalkylene —(CH₂)_(n)OC₃₋₆ alkenylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)C₁₋₆ —(CH₂)_(n)C(O)C₂₋₆ —(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)OC₁₋₆ —(CH₂)_(n)C(O)OC₂₋₆ —(CH₂)_(n)C(O)O— —(CH₂)_(n)C(O)OC₃₋₆ alkylene alkenylene C₃₋₆ cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC(O)C₁₋₆ —(CH₂)_(n)OC(O)C₂₋₆ —(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₁₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)C₁₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)NR²⁰C₁₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—C₁₋₆ —(CH₂)_(n)—S—C₂₋₆ —(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— C₁₋₆ alkylene C₂₋₆ alkenylene C₃₋₆ cycloalkylene C₃₋₆ cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO₂—C₁₋₆ —(CH₂)_(n)—SO₂—C₂₋₆ —(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)—SO₂—C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)— (CH₂)_(n)C(O)(CH₂)_(n)—SO₂— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— SO₂— SO₂— C₃₋₆ cycloalkylene C₃₋₆ cycloalkenylene C₁₋₆ alkylene C₂₋₆ alkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO—C₁₋₆ —(CH₂)_(n)—SO—C₂₋₆ —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO— —(CH₂)_(n)C(O)(CH₂)_(n)—SO— SO— SO— C₃₋₆ cycloalkenylene C₃₋₆ cycloalkenylene C₁₋₆ alkylene C₂₋₆ alkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—S—C₁₋₆ —(CH₂)_(n)—S—S—C₂₋₆ —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₃₋₆ alkylene alkenylene cycloalkylene cycloalkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— S— S— C₃₋₆ cycloalkylene C₃₋₆ cycloalkenylene C₁₋₆ alkylene C₂₋₆ alkenylene optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C₁₋₆ alkylene- —(CH₂)_(n)C₂₋₆ —(CH₂)_(n)C₃₋₆ cycloalkylene- —(CH₂)_(n)C₃₋₆ NR²¹— alkenylene-NR²¹— NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC₁₋₆ alkylene- —(CH₂)_(n)OC₂₋₆ —(CH₂)_(n)OC₃₋₆ cycloalkylene- —(CH₂)_(n)OC₃₋₆ NR²¹— alkenylene-NR²¹— NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)C₁₋₆ —(CH₂)_(n)C(O)C₂₋₆ —(CH₂)_(n)C(O)C₃₋₋₆ —(CH₂)_(n)C(O)C₃₋₆ alkylene-NR²¹— alkenylene-NR²¹— cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)OC₁₋₆ —(CH₂)_(n)C(O)OC₂₋₆ —(CH₂)_(n)C(O)O—C₃₋₋₆ —(CH₂)_(n)C(O)OC₃₋₆ alkylene-NR²¹— alkenylene-NR²¹— cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC(O)C₁₋₆ —(CH₂)_(n)OC(O)C₂₋₆ —(CH₂)_(n)OC(O)—C₃₋₋₆ —(CH₂)_(n)OC(O)C₃₋₆ alkylene-NR²¹— alkenylene-NR²¹— cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₁₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ alkylene-NR²¹— alkenylene-NR²¹— cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)C₁₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆ —(CH₂)_(n)NR²⁰C(O)— —(CH₂)_(n)NR²⁰C(O)— alkylene-NR²¹— alkenylene-NR²¹— C₃₋₆ cycloalkylene-NR²¹— C₃₋₆ cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)NR²⁰C₁₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ alkylene-NR²¹— alkenylene-NR²¹— cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—C₁₋₆ —(CH₂)_(n)—S—C₂₋₆ —(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₃₋₆ alkylene-NR²¹— alkenylene cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— C₁₋₆ alkylene-NR²¹— C₂₋₆ alkenylene-NR²¹— C₃₋₆ cycloalkylene-NR²¹— C₃₋₆ cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO₂—C₁₋₆ —(CH₂)_(n)—SO₂—C₁₋₆ —(CH₂)_(n)—SO₂— —(CH₂)_(n)—SO₂—C₃₋₆ alkylene-NR²¹— alkenylene-NR²¹— C₃₋₆ cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— SO₂— SO₂— C₃₋₆ cycloalkylene-NR²¹— C₃₋₆ cycloalkenylene-NR²¹— C₁₋₆ alkylene-NR²¹— C₂₋₆ alkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO—C₁₋₆ —(CH₂)_(n)—SO—C₂₋₆ —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₃₋₆ alkylene-NR²¹— alkenylene-NR²¹— cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO— —(CH₂)_(n)C(O)(CH₂)_(n)—SO— SO— SO— C₃₋₆ cycloalkylene-NR²¹— C₃₋₆ cycloalkenylene-NR²¹— C₁₋₆ alkylene-NR²¹— C₂₋₆ alkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—S—C₁₋₆ —(CH₂)_(n)—S—S—C₂₋₆ —(CH₂)_(n)—S—S— —(CH₂)_(n)—S—S—C₃₋₆ alkylene-NR²¹— alkenylene-NR²¹— C₃₋₆ cycloalkylene-NR²¹— cycloalkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— S— S— C₃₋₆ cycloalkylene-NR²¹— C₃₋₆ cycloalkenylene-NR²¹— C₁₋₆ alkylene-NR²¹— C₂₋₆ alkenylene-NR²¹— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C₁₋₆ —(CH₂)_(n)C₂₋₆ —(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)-— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC₁₋₆ —(CH₂)_(n)OC₂₋₆ —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)C₁₋₆ —(CH₂)_(n)C(O)C₂₋₆ —(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)-— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)OC₁₋₆ —(CH₂)_(n)C(O)OC₂₋₆ —(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)OC₃₋₆ alkylene-(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC(O)C₁₋₆ —(CH₂)_(n)OC(O)C₂₋₆ —(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₁₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)C₁₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₁₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)NR²⁰C₁₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—C₁₋₆ —(CH₂)_(n)—S—C₂₋₆ —(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— C₁₋₆ alkylene-C(O) C₂₋₆ alkenylene-C(O) C₃₋₆ cycloalkylene-C(O) C₃₋₆ cycloalkenylene-C(O) optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO₂—C₁₋₆ —(CH₂)_(n)—SO₂—C₂₋₆ —(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)—SO₂—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— SO₂—C₁₋₆ alkylene-C(O) C₂₋₆ alkenylene-C(O) C₃₋₆ cycloalkylene-C(O) C₃₋₆ cycloalkenylene-C(O) optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO—C₁₋₆ —(CH₂)_(n)—SO—C₂₋₆ —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO— —(CH₂)_(n)—C(O)(CH₂)_(n)—SO— —(CH₂)_(n)—C(O)(CH₂)_(n)—SO— SO—C₁₋₆ alkylene—C(O) C₂₋₆ alkenylene-C(O) C₃₋₆ cycloalkylene-C(O) C₃₋₆ cycloalkenylene-C(O) optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—S—C₁₋₆ —(CH₂)_(n)—S—S—C₂₋₆ —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₃₋₆ alkylene-C(O)— alkenylene-C(O)— cycloalkylene-C(O)— cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— C₁₋₆ alkylene-C(O) C₂₋₆ alkenylene-C(O) C₃₋₆ cycloalkylene-C(O) C₃₋₆ cycloalkenylene-C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)OC₁₋₆ —NR²⁰C(O)(CH₂)_(n)O—C₂₋₆ —NR²⁰C(O)(CH₂)_(n)O—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)O—C₃₋₆ alkylene-(CO) alkenylene-(CO) cycloalkylene-(CO) cycloalkenylene-(CO) optionally substituted optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)—S— NR²⁰C(O)(CH₂)_(n)—S— —NR²⁰C(O)(CH₂)_(n)—S— —NR²⁰C(O)(CH₂)_(n)—S— C₁₋₆ alkylene-(CO) C₂₋₆ alkenylene-(CO) C₃₋₆ cycloalkylene-(CO) C₃₋₆ cycloalkenylene-(CO) optionally substituted optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)NR²¹ —NR²⁰C(O)(CH₂)_(n)NR²¹— —NR²⁰C(O)(CH₂)_(n)NR²¹— —NR²⁰C(O)(CH₂)_(n)NR²¹— C₁₋₆ alkylene-(CO) C₂₋₆ alkenylene-(CO) C₃₋₆ cycloalkylene-(CO) C₃₋₆ cycloalkenylene-(CO) optionally substituted optionally substituted optionally substituted optionally substituted C(O)NR²⁰(CH₂)_(n)OC₁₋₆ —C(O)NR²⁰(CH₂)_(n)O—C₂₋₆ —C(O)NR²⁰(CH₂)_(n)O—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)O—C₃₋₆ alkylene-(CO) alkenylene-(CO) cycloalkylene-(CO) cycloalkenylene-(CO) optionally substituted optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)—S— —C(O)NR²⁰(CH₂)_(n)—S— —C(O)NR²⁰(CH₂)_(n)—S— —C(O)NR²⁰(CH₂)_(n)—S— C₁₋₆ alkylene-(CO) C₂₋₆ alkenylene-(CO) C₃₋₆ cycloalkylene-(CO) C₃₋₆ cycloalkenylene-(CO) optionally substituted optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)— vC(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)— NR²¹C₁₋₆ alkylene-(CO) NR²¹ C₂₋₆ alkenylene-(CO) NR²¹ C₃₋₆ cycloalkylene-(CO) NR²¹ C₃₋₆ cycloalkenylene-(CO) optionally substituted optionally substituted optionally substituted optionally substituted —C(O)(CH₂)_(n)C₁₋₆ alkylene —C(O)(CH₂)_(n)C₁₋₆ alkenylene —C(O)(CH₂)_(n)C₃₋₆ cycloalkylene —C(O)(CH₂)_(n)C₃₋₆ —(CH₂)_(n)— —(CH₂)_(n)— —(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionally substituted optionally substituted optionally substituted optionally substituted —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₃₋₆ —C(O)O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)— alkenylene-(CH₂)_(n)— cycloalkylene (CH₂)_(n)— cycloalkenylene (CH₂)_(n)— optionally substituted optionally substituted optionally substituted optionally substituted —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₃₋₆ —C(O)O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O— cycloalkylene (CH₂)_(n)—O— cycloalkenylene (CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted optionally substituted —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₃₋₆ —C(O)O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O— cycloalkylene (CH₂)_(n)—O— cycloalkenylene (CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted optionally substituted —C(O)(CH₂)_(n)C₁₋₆ —C(O)(CH₂)_(n)C₁₋₆ —C(O)(CH₂)_(n)C₃₋₆ —C(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)— cycloalkylene (CH₂)_(n)—C(O)— cycloalkenylene (CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₁₋₆ —C(O)O(CH₂)_(n)C₃₋₆ —C(O)O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)— cycloalkylene (CH₂)_(n)—C(O)— cycloalkenylene (CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)— alkenylene-(CH₂)_(n)— cycloalkylene-(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionally substituted optionally substituted optionally substituted optionally substituted —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₃₋₆ —O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)— alkenylene-(CH₂)_(n)— cycloalkylene-(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionally substituted optionally substituted optionally substituted optionally substituted —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O— cycloalkylene-(CH₂)_(n)—O— cycloalkenylene-(CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted optionally substituted —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₃₋₆ —O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O— cycloalkylene-(CH₂)_(n)—O— cycloalkenylene-(CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted optionally substituted —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₁₋₆ —OC(O)(CH₂)_(n)C₃ ₆ —OC(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)— cycloalkylene-(CH₂)_(n)—C(O)— cycloalkenylene-(CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₁₋₆ —O(CH₂)_(n)C₃₋₆ —O(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)— cycloalkylene-(CH₂)_(n)—C(O)— cycloalkenylene-(CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)C₃₋₆ C₁₋₆ alkylene-(CH₂)_(n)— C₁₋₆ alkenylene-(CH₂)_(n)— C₃₋₆ cycloalkylene-(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionally substituted optionally substituted optionally substituted optionally substituted NR²⁰C(O)(CH₂)_(n)— —NR²⁰C(O)(CH₂)_(n)— —NR²⁰C(O)(CH₂)_(n)— —NR²⁰C(O)(CH₂)_(n)C₃₋₆ C₁₋₆ alkylene-(CH₂)_(n)— C₁₋₆ alkenylene-(CH₂)_(n)— C₃₋₆ cycloalkylene-(CH₂)_(n)— cycloalkenylene-(CH₂)_(n)— optionally substituted optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n) —C(O)NR²⁰(CH₂)_(n) —C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)C₃₋₆ C₁₋₆ alkylene-(CH₂)_(n)—O— C₁₋₆ alkenylene-(CH₂)_(n)—O— C₃₋₆ cycloalkylene-(CH₂)_(n)—O— cycloalkenylene-(CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)C₁₋₆ —NR²⁰C(O)(CH₂)_(n)C₁₋₆ —NR²⁰C(O)(CH₂)_(n)— —NR²⁰C(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—O— alkenylene-(CH₂)_(n)—O— C₃₋₆ cycloalkylene-(CH₂)_(n)—O— cycloalkenylene-(CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)C₁₋₆ —C(O)NR²⁰(CH₂)_(n)C₁₋₆ —C(O)NR²⁰(CH₂)_(n)— —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)— C₃₋₆ cycloalkylene-(CH₂)_(n)—C(O)— cycloalkenylene-(CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)C₁₋₆ —NR²⁰C(O)(CH₂)_(n)C₁₋₆ —NR²⁰C(O)(CH₂)_(n)— —NR²⁰C(O)(CH₂)_(n)C₃₋₆ alkylene-(CH₂)_(n)—C(O)— alkenylene-(CH₂)_(n)—C(O)— C₃₋₆ cycloalkylene-(CH₂)_(n)—C(O)— cycloalkenylene-(CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₃₋₆ alkynylene heterocycloalkylene heterocycloalkenylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₂₋₆ heterocycloalkylene heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₂₋₆ heterocycloalkylene heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)OC₃₋₆ —(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)OC₂₋₆ heterocycloalkylene heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC(O)C₃₋₆ —(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₂₋₆ heterocycloalkylene heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰—C₃₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ heterocycloalkylene heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆ heterocycloalkylene heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)NR²⁰— —(CH₂)_(n)C(O)NR²⁰— —(CH₂)_(n)C(O)NR²⁰— optionally substituted optionally substituted C₂₋₆ alkynylene C₃₋₆ heterocycloalkylene C₃₋₆ heterocycloalkenylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S— —(CH₂)_(n)—S— —(CH₂)_(n)—S—C₂₋₆ C₃₋₆ C₃₋₆ alkynylene heterocycloalkylene heterocycloalkenylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— C₃₋₆ heterocycloalkylene C₃₋₆ heterocycloalkenylene C₂₋₆ alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)—SO₂— —(CH₂)_(n)—SO₂—C₂₋₆ heterocycloalkylene C₃₋₆ heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂— SO₂— SO₂— C₂₋₆ alkynylene C₃₋₆ heterocycloalkylene C₃₋₆ heterocycloalkenylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)— —(CH₂)_(n)C(O)(CH₂)_(n)—SO— SO— SO— C₂₋₆ alkynylene C₃₋₆ heterocycloalkylene C₃₋₆ heterocycloalkenylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene heterocycloalkenylene alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S— —(CH₂)_(n)C(O)(CH₂)_(n)—S—S— S— S— C₂₋₆ alkynylene C₃₋₆ heterocycloalkylene C₃₋₆ heterocycloalkenylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)₃₋₆ —(CH₂)_(n)C(O)—C₃₋₆ —(CH₂)_(n)C(O)C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)OC₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)—SO₂—C₁₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—C(O)(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene-NR²¹— heterocycloalkenylene-NR²¹— alkynylene-NR²¹— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₃₋₆ —(CH₂)_(n)C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₃₋₆ —(CH₂)_(n)OC₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₃₋₆ —(CH₂)_(n)C(O)C₃₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)OC₃₋₆ —(CH₂)_(n)C(O)O—C₃₋₆ —(CH₂)_(n)C(O)OC₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)OC(O)C₃₋₆ —(CH₂)_(n)OC(O)—C₃₋₆ —(CH₂)_(n)OC(O)C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰—C₃₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ heteroalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C(O)C₃₋₆ —(CH₂)_(n)NR²⁰C(O)—C₃₋₆ —(CH₂)_(n)NR²⁰C(O)C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)NR²⁰C₃₋₆ —(CH₂)_(n)NR²⁰—C₃₋₆ —(CH₂)_(n)NR²⁰C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)NR²⁰C₃₋₆ —(CH₂)_(n)C(O)NR²⁰—C₃₋₆ —(CH₂)_(n)C(O)NR²⁰C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-C(O) heterocycloalkenylene-C(O) alkynylene-C(O) optionally substituted optionally substituted optionally substituted —(CH₂)_(n)SO₂—C₃₋₆ —(CH₂)_(n)SO₂—C₃₋₆ —(CH₂)_(n)SO₂—C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO₂—C₂₋₆ heterocycloalkylene-C(O) heterocycloalkenylene-C(O) alkynylene-C(O) optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—SO—C₂₋₆ heterocycloalkylene-C(O) heterocycloalkenylene-C(O) alkynylene-C(O) optionally substituted optionally substituted optionally substituted —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene-C(O)— heterocycloalkenylene-C(O)— alkynylene-C(O)— optionally substituted optionally substituted optionally substituted —(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₃₋₆ —(CH₂)_(n)C(O)(CH₂)_(n)—S—S—C₂₋₆ heterocycloalkylene-C(O) heterocycloalkenylene-C(O) alkynylene-C(O) optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)O—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)O—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)O—C₂₋₆ heterocycloalkylene-C(O) heterocycloalkenylene-C(O) alkynylene-C(O) optionally substituted optionally substituted optionally substituted NR²⁰C(O)(CH₂)_(n)—S—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—S—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-C(O) heterocycloalkenylene-C(O) alkynylene-C(O) optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)NR²¹—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)NR²¹—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)NR²¹—C₂₋₆ heterocycloalkylene-C(O) heterocycloalkenylene-C(O) alkynylene-C(O) optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)O—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)O—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)O—C₂₋₆ heterocycloalkylene-(CO) heterocycloalkenylene-(CO) alkynylene-(CO) optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)—S—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—S—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—S—C₂₋₆ heterocycloalkylene-(CO) heterocycloalkenylene-(CO) alkynylene-(CO) optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)—NR²¹—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—NR²¹—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—NR²¹C₂₋₆ heterocycloalkylene-(CO) heterocycloalkenylene-(CO) alkynylene-(CO) optionally substituted optionally substituted optionally substituted —C(O)(CH₂)_(n)C₃₋₆ —C(O)(CH₂)_(n)C₃₋₆ —C(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene-(CH₂)_(n)— heterocycloalkenylene-(CH₂)_(n)— alkynylene-(CH₂)_(n)— optionally substituted optionally substituted optionally substituted —C(O)O(CH₂)_(n)—C₃₋₆ —C(O)O(CH₂)_(n)—C₃₋₆ —C(O)O(CH₂)_(n)C₁₋₆ heterocycloalkylene-(CH₂)_(n)— heterocycloalkenylene-(CH₂)_(n)— alkynylene-(CH₂)_(n)— optionally substituted optionally substituted optionally substituted —C(O)(CH₂)_(n)—C₃₋₆ —C(O)(CH₂)_(n)—C₃₋₆ —C(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted —C(O)O(CH₂)_(n)—C₃₋₆ —C(O)O(CH₂)_(n)—C₃₋₆ —C(O)O(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted —C(O)(CH₂)_(n)C₃₋₆ —C(O)(CH₂)_(n)—C₃₋₆ —C(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted —C(O)(CH₂)_(n)C₃₋₆ —C(O)(CH₂)_(n)—C₃₋₆ —C(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted —OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)—C₃₋₆ —OC(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n) (CH₂)_(n) (CH₂)_(n) optionally substituted optionally substituted optionally substituted —O(CH₂)_(n)—C₃₋₆ —O(CH₂)_(n)—C₃₋₆ —O(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n) (CH₂)_(n) (CH₂)_(n) optionally substituted optionally substituted optionally substituted —OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)—C₃₋₆ —OC(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted —O(CH₂)_(n)C₃₋₆ —O(CH₂)_(n)C₃₋₆ —O(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted —OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)C₃₋₆ —OC(O)(CH₂)_(n)C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted —O(CH₂)_(n)—C₃₋₆ —O(CH₂)_(n)—C₃₋₆ —O(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)— (CH₂)_(n)— (CH₂)_(n)— optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ NR²⁰C(O)(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)— (CH₂)_(n)— (CH₂)_(n)— optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—O— (CH₂)_(n)—O— (CH₂)_(n)—O— optionally substituted optionally substituted optionally substituted —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₃₋₆ —C(O)NR²⁰(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— optionally substituted optionally substituted optionally substituted —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₃₋₆ —NR²⁰C(O)(CH₂)_(n)—C₁₋₆ heterocycloalkylene- heterocycloalkenylene- alkynylene-(CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)— (CH₂)_(n)—C(O)—

-   -   Each R²⁰ and R²¹ is independently selected from the group         consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl,         arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴ are each independently hydrogen or alkyl.

In some embodiments, the FKBD-containing moiety before incorporated into the macrocycle can have a structure according to Formula (III) or an optically pure stereoisomer or pharmaceutically acceptable salt thereof.

Wherein L is selected from the structure in Table 1; A is CH₂, NH, 0, or S; each X is independently O, NH, or NMe; E is CH or N;

represents a single or a double bond. n is an integer selected from 0 to 4.

Each R¹ is selected from the group consisting of H, halogen, hydroxyl, C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, and CO₂C₁₋₂₀alkyl. R² is selected from the group consisting of H, halogen, hydroxyl, C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, and CO₂C₁₋₂₀alkyl. R³ is selected from the group consisting of C₆₋₁₅aryl and C₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

V is

Z is a bond,

wherein R⁴ and R⁵ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR⁶, CR⁶, N, and NR⁶, wherein R⁶ is hydrogen or alkyl.

In some embodiments, the FKBD-containing moiety before incorporated into the macrocycle can have a structure according to Formula (IV) or an optically pure stereoisomer or pharmaceutically acceptable salt thereof.

Wherein L is selected from the structures in Table 1; A is CH₂, NH, O, or S; each X is independently O or NH; E is CH or N; each R¹ is selected from the group consisting of H, halogen, hydroxyl, C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, and CO₂C₁₋₂₀alkyl; each R² is selected from the group consisting of H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl; n is an integer selected from 0 to 4; and m is an integer selected from 0 to 5.

In some embodiments, the Rapafucin compounds in the present disclosure can have a structure according to Formula (V) or an optically pure stereoisomer or pharmaceutically acceptable salt thereof.

Wherein L is selected from the groups in Table 1; A is CH₂, NH, NMe, O, S(O)₂ or S; each X is independently O, NMe, or NH; E is CH or N.

Each of R¹, R², R³, and R⁴ can be independently selected from the group consisting of H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, CO₂C₁₋₂₀alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₁₋₁₀alkoxy, C₆₋₁₅aryl, C₆₋₁₅aryloxy, C₆₋₁₅arylthio, C₂₋₁₀carboxyl, C₁₋₁₀alkylamino, thiol, C₁₋₁₀alkylthio, C₁₋₁₀alkyldisulfide, C₆₋₁₅arylthio, C₁₋₁₀heteroarylthio, (C₃₋₈cycloalkyl)thio, C₂₋₁₀heterocyclylthio, sulfonyl, C₁₋₁₀alkylsulfonyl, amido, C₁₋₁₀alkylamido, selenol, C₁₋₁₀alkylselenol, C₆₋₁₅arylselenol, C₁₋₁₀heteroarylselenol, (C₃₋₈cycloalkyl)selenol, C₂₋₁₀heterocyclylselenol, guanidino, C₁₋₁₀alkylguanidino, urea, C₁₋₁₀alkylurea, ammonium, C₁₋₁₀alkylammonium, cyano, C₁₋₁₀alkylcyano, C₁₋₁₀alkylnitro, adamantine, phosphonate, C₁₋₁₀alkylphosphonate, and C₆₋₁₅arylphosphonate, each of the above can be optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₂₀alkyl, substituted C₁₋₂₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

Or any R⁴ forms a cyclic structure formed with any R³, the cyclic structure is selected from the group consisting of C₂₋₁₀heterocyclyl and C₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅5arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

n is an integer selected from 0 to 4; m is an integer selected from 0 to 5; each p is an integer independently selected from 0 to 2; q is an integer selected from 1 to 10.

In some embodiments, q can be 1. In some embodiments, q can be 2. In some embodiments, q can be 3. In some embodiments, q can be 4. In some embodiments, q can be 5. In some embodiments, q can be 6. In some embodiments, q can be 7. In some embodiments, q can be 8. In some embodiments, q can be 9. In some embodiments, q can be 10. In specific embodiments, q is 3 or 4.

In some embodiments, the Rapafucin compounds in the present disclosure can have a structure according to Formula (VI) or an optically pure stereoisomer or pharmaceutically acceptable salt thereof.

Each L¹, L², or L³ can be independently selected from the linker structures in Table 1. Each AA₁, AA₂, AA₃, or AA₄ can be independently selected from the amino acid monomers shown in Table 3 below. X can be CH₂, NH, O, or S; Y can be O, NH, or N-alkyl; E can be CH or N; n is an integer selected from 0 to 4. Amino acids can be either N—C linked or C—N linked.

Each R¹ is selected from the group consisting of H, halogen, hydroxyl, C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, and CO₂C₁₋₂₀alkyl. R² is selected from the group consisting of C₆₋₁₅aryl and C₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

V is

Z is a bond,

wherein R³ and R⁴ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR⁵, CR⁵, N, and NR⁵, wherein R⁵ is hydrogen or alkyl.

Synthetic route to Rapafucins. There are several methods for the synthesis of rapafucins including both solid and solution phase synthesis. These methods can result in modifications to the linker(s) and/or the effector domain which include alkylations, amide bond formations, double bond metathesis, oxadiazole formation, triazole formations, dithiol formations, sulfone formations, Diels-Alder cycloadditions, and others.

We applied solid-phase peptide synthesis to assemble the polypeptide effector domains. The pre-assembled FKBD capped with a carboxylic acid at one end and an olefin at the other was subsequently coupled to the polypeptide that remained tethered on beads. To facilitate purification of the newly formed macrocycles, we adopted a coupled macrocyclization and cyclative release strategy whereby the macrocyclization is accompanied by the concurrent release of the macrocyclic products from the solid beads. One skilled in the art can contemplate different macrocyclization methods for the synthesis of Rapafucin molecules in the present disclosure. In some embodiments, a ring-closing metathesis/cyclative release (RCM) is used. In some embodiments, macrolactamization can be used for efficient parallel synthesis of different Rapafucins. A cis-C6 linker can be used for construction of Rapafucin libraries. A combination of medium temperature and catalyst loading (140° C., 30 mol % Hoveyda-Grubbs II catalyst) for the ensuing large-scale synthesis of Rapafucin libraries.

Other ring-closing methods can be used to synthesize the Rapafucin molecules disclosed herein. Exemplary methods can include, but not limited to aminolysis, chemoenzymatic method, click chemistry, macrocylization through ring contraction using auxiliary groups, macrocylization mediated through sulfur containing groups, macrocylization via cycloaddition, macrocylization via Wittiga or Wittig like reactions, macrocylization from multicomponent reactions, metal-assisted macrocylization, macrocylization through C—N bond formation, macrocylization through C—O bond formation, alkylation with or without metal assistance, intramolecular cyclopropanation, oxidative coupling of arenes, side chain cyclization, and oxidative coupling of arenes. Each of these macrocyclization method can be conducted in solid phase or solution phase. The macrocyclization reactions through ring contraction using auxiliary groups can include, but not limited to using hydroxyl benzaldehyde, using hydroxyl nitro phenol, and using nitro vinyl phenol. The macrocylization reactions mediated through sulfur containing groups can include, but not limited to thiazolidine formation O to N acyl transfer, transesterification S to N acyl transfer, ring chain tautomerization S to N acyl transfer, Staudinger ligation ring contraction, bis-thiol-ene macrocyclization, thiol-ene macrocyclization, thiolalkylation, and disulfide formation. The macrocyclization reactions via cycloaddtion can include, but not limited to phosphorene-azide ligation and oxadiazole graft. Metal assisted macrocyclization can include, but not limited to C—C bond formation, Suzuki coupling, Sonogashira coupling, Tasuji-Trost reaction, Glaser-Hay coupling, and Nickel catalyzed macrocylication. Macrocyclization reactions via C—N bond formation can include, but not limited to Ullmann coupling and Buchwald-Hartwig animation. Macrocyclization reactions via C—O bond formation can include, but not limited to Chan-Lam-Evans coupling, C—H activation, and Ullmann coupling. Macrocyclization reactions via alkylation can include enolate chemistry, Williamson etherification, Mitsunobu reaction, aromatic nucleophilic substitution (SNAr), and Friedel-Crafts type alkylation.

In some embodiments, Rapafucin molecules can be cyclized using the methods described in Marsault, E., & Peterson, M. L. (Eds.). (2017). Practical Medicinal Chemistry with Macrocycles: Design, Synthesis, and Case Studies, which is hereby incorporate d by reference in its entirety. Some non-limiting examples of the macrocyclization methods are shown in Table 2 below, each n can be independently an integer selected from 0 to 10.

TABLE 2 Additional macrocyclization methods that can be used for Rapafucin synthesis. Macrocyclization reactions Reaction scheme Cyclization by intramolecular aminolysis

Macrocyclization via Chemoenzymatic methods

Cyclization by intramolecular aminolysis-II

Macrocyclization through ring contraction using auxiliary groups- using hydroxyl benzaldehyde

Macrocyclization through ring contraction using auxiliary groups- using hydroxyl nitro phenol

Macrocyclization through ring contraction using auxiliary groups- using nitro vinyl phenol

Macrocyclization mediated through sulfur containing groups-via thiazolidine formation O to N acyl transfer

Macrocyclization mediated through sulfur containing groups-via transesterification S to N acyl transfer

Macrocyclization mediated through sulfur containing groups-via ring chain tautomerization S to N acyl transfer

Macrocyclization mediated through sulfur containing groups- Staudinger ligation ring contraction

Macrocyclization mediated through sulfur containing groups-bis-thiol-ene macrocyclization

Macrocyclization mediated through sulfur containing groups-thiol-ene macrocyclization

Macrocyclization mediated through sulfur containing groups- thioalkylation

Macrocyclization mediated through sulfur containing groups-disulfide formation

Macrocyclization via cycloaddition- phosphorene-azide ligation

Macrocyclization via azide-alkyne cycloaddition 1,3-dipolar Huisgen cycloaddition

Macrocyclization via cycloaddition- oxadiazole graft using (N- isocyanimino) triphenylphosphorane

Macrocyclization via Wittig or Horner- Wadsworth- Emmons or Masamune-Roush reactions or Still- Gennari olefination

Macrocyclization from multicomponent reactions

Metal assisted macrocyclization- C—C bond formation (Metals include Pd, Ni, Cu, Ru, or Au)

Metal assisted macrocyclization C═C bond formation (Metals include Pd, Ni, Cu, Ru, or Au)

Metal assisted macrocylization- Suzuki coupling

Metal assisted macrocylization- Sonogashira coupling

Metal assisted macrocylization- Tsuji-Trost reaction

Metal assisted macrocylization- Glaser-Hay coupling

Metal assisted macrocylization- Nickel catalyzed macrocyclization

Macrocyclization via C—N bond formation- Ullmann coupling

Macrocyclization via C—N bond formation- Buchwald-Hartwig amination

Macrocyclization via C—N bond formation- Chan-Lam-Evans coupling

Macrocyclization via C—N bond formation-C—H activation

Macrocyclization via C—N bond formation- Ullmann coupling

Macrocyclization via alkylation- enolate chemistry

Macrocyclization via alkylation- Williamson etherification

Macrocyclization via alkylation- Mitsunobu reaction

Macrocyclization via alkylation- aromatic nucleophilic substitution (SNAr)

Macrocyclization via alkylation- Friedel-Crafts type alkylations

Macrocyclization through intramolecular cyclopropanation

Macrocyclization through oxidative coupling of Arenes

Macrocyclization- side chain cyclization

Macrocyclization- oxidative coupling of arenes

In some embodiments, the Rapafucin compounds in the present disclosure can have a structure according to Formula (VII) or an optically pure stereoisomer or pharmaceutically acceptable salt thereof.

Each T₁ or T₂ can be independently selected from the terminal structures as outlined in Table 2 above before macrocyclization. Each L₁, L₂, or L₃ can be independently selected from the linker structures in Table 1. Each AA can be independently selected from the amino acid monomers shown in Table 3 below. X can be CH₂, NH, O, or S; Y can be O, NH, or N-alkyl; E can be CH or N; n is an integer selected from 0 to 4. Amino acids can be either N—C linked or C—N linked.

In some embodiments, m can be 1. In some embodiments, m can be 2. In some embodiments, m can be 3. In some embodiments, m can be 4. In some embodiments, m can be 5. In some embodiments, m can be 6. In some embodiments, m can be 7. In some embodiments, m can be 8. In some embodiments, m can be 9. In some embodiments, m can be 10. In a specific embodiment, m is 3 or 4.

V is

Z is a bond,

wherein R³ and R⁴ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR⁵, CR⁵, N, and NR⁵, wherein R⁵ is hydrogen or alkyl.

Each R¹ is selected from the group consisting of H, halogen, hydroxyl, C₁₋₂₀ alkyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, and CO₂C₁₋₂₀alkyl. R² is selected from the group consisting of C₆₋₁₅aryl and C₁₋₁₀heteroaryl optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl.

Table 3 below shows the FKBD moieties with linkers before incorporated into the Rapafucin macrocylic structure.

TABLE 3 The FKBD/linker moieties used in the present disclosure. FKBD identifier Chemical Structure aFKBD

eFKBD

Raa1

Raa2

Raa3

Raa4

Raa5

Raa6

Raa7

Raa8

Raa9

Raa10

Raa11

Raa12

Raa13

Raa14

Raa15

Raa16

Raa17

Raa18

Raa19

Raa20

Raa21

Raa22

Raa25

Raa26

Raa27

Raa28

Raa29

Raa30

Rae1

Rae2

Rae3

Rae4

Rae5

Rae9

Rae10

Rae11

Rae12

Rae13

Rae14

Rae15

Rae16

Rae17

Rae18

Rae19

Rae20

Rae21

Rae22

Rae23

Rae24

Rae25

Rae26

Rae27

Rae28

Rae29

Rae30

Rae31*

Rae32

Rae33

Rae34

Rae35

Rae36

Rae37

Rae38

*This FKBD is reduced and cyclized via lactamization.

Table 4 below shows the amino acid monomers used for the the Rapafucin macrocylic compounds synthesis in the present disclosure.

TABLE 4 The monomers used in the present disclosure. Entry Monomer No. identifier Chemical Structure  1 G

 2 Sar

 3 dA

 4 A

 5 bAla

 6 Dpr

 7 ra199

 8 mA

 9 Alb

 10 Abu

 11 C

 12 dC

 13 SeC

 14 DSec

 15 dS

 16 S

 17 ra165

 18 Aze

 19 ra126

 20 ra524

 21 dP

 22 P

 23 ra132

 24 SbPro

 25 RbPro

 26 ra603

 27 Dab

 28 ra484

 29 ra203

 30 ra201

 31 ra202

 32 isoV

 33 ra130

 34 Nva

 35 ra131

 36 dV

 37 V

 38 bVal

 39 Hcy

 40 mC

 41 dT

 42 T

 43 mS

 44 Hse

 45 Bux

 46 Om

 47 dN

 48 N

 49 RbAsn

 50 SbAsn

 51 RbAsp & dD

 52 D

 53 ra344

 54 mV

 55 ra345

 56 ra379

 57 ra359

 58 Nle

 59 Dl

 60 L

 61 dI

 62 I

 63 Tle

 64 RbIle

 65 SbIle

 66 SbLeu

 67 RbLeu

 68 ra74

 69 RbMet

 70 SbMet

 71 M

 72 dM

 73 Pen

 74 ra371

 75 mT

 76 ra582

 77 ra380

 78 ra473

 79 ra341

 80 ra538

 81 ra555

 82 ra550

 83 Spg

 84 ra144

 85 ra189

 86 ra330

 87 ra541

 88 ra528

 89 ra168

 90 ra532

 91 Roh4P

 92 ra508

 93 ra557

 94 ra576

 95 Glp

 96 ra505

 97 ra518

 98 ra584

 99 ra372

100 ra83

101 ra162

102 ra169

103 ra127

104 ra76

105 ra600

106 ra128

107 ra564

108 ra510

109 ra464

110 ra466

111 ra543

112 ra170

113 m4oh3P

114 dK

115 K

116 SbLys

117 RbLys

118 mN

119 dQ

120 Q

121 RbGln

122 SbGln

123 mD

124 dE

125 E

126 ra206

127 RbGlu

128 mI

129 ra352

130 ra147

131 ra207

132 mL

133 ra530

134 Elscy

135 mM

136 ra61

137 Cya

138 ra401

139 mK

140 oh5K

141 mQ

142 mE

143 Aad

144 ra458

145 ra459

146 ra583

147 ra310

148 ra563

149 Tza

150 ra301

151 ra507

152 ra509

153 ra602

154 ra601

155 Phg

156 ra84

157 ra337

158 ra338

159 ra363

160 ra364

161 Thl

162 ra368

163 ra67

164 ra68

165 dH

166 H

167 SbHis

168 RbHis

169 ra405

170 ra90

171 ra406

172 ra89

173 ra91

174 ra176

175 ra462

176 ra461

177 ra565

178 ra122

179 dF

180 F

181 ra527

182 Cha

183 SbPhe

184 RbPhe

185 ra516

186 ra325

187 ra450

188 ra522

189 mH

190 Hhs

191 ra490

192 ra609

193 ra173

194 ra102

195 ra542

196 Olc

197 ra540

198 dR

199 R

200 RbArg

201 SbArg

202 Apm

203 ra355

204 ra300

205 ra581

206 ra142

207 ra183

208 ra562

209 Sta

210 Cit

211 mR

212 Har

213 ra664

214 Dpm

215 m3K

216 Ra590

217 ra307

218 ra547

219 Asu

220 ra535

221 ra348

222 Aca

223 Gla

224 ra80

225 ra545

226 Tic

227 ra351

228 ra350

229 ra69

230 ra101

231 ra204

232 ra521

233 ra523

234 ra172

235 ra195

236 mF

237 ra558

238 ra120

239 ra659

240 ra134

241 ra59

242 ra549

243 ra104

244 ra123

245 ra87

246 ra336

247 ra116

248 ra665

249 ra117

250 ra115

251 ra118

252 ra339

253 ra119

254 ra666

255 ra121

256 ra551

257 ra539

258 ra381

259 dY

260 Y

261 ra469

262 ra400

263 ra106

264 ra335

265 ra513

266 ra329

267 SbTyr

268 RbTyr

269 ra658

270 ra113

271 ra114

272 ra596

273 ra112

274 ra561

275 ra208

276 ra63

277 ra66

278 ra55

279 ra62

280 ra59

281 ra534

282 ra387

283 ra386

284 ra374

285 ra360

286 ra64

287 ra65

288 ra382

289 ra537

290 ra88

291 ra209

292 ra497

293 ra185

294 mY

295 ra133

296 ra667

297 ra124

298 Uraal

299 ra594

300 Dsu

301 ra456

302 ra457

303 ra589

304 ra559

305 ra536

306 ra548

307 ra573

308 ra86

309 ra574

310 ra533

311 ra75

312 ra105

313 ra136

314 ra454

315 ra321

316 ra588

317 ra560

318 ra517

319 ra648

320 ra317

321 ra302

322 ra660

323 ra108

324 ra378

325 ra109

326 ra597

327 ra111

328 ra579

329 App

330 Cap

331 dW

332 W

333 SbTrp

334 RbTrp

335 ra347

336 ra575

337 ra404

338 ra407

339 ra129

340 ra608

341 ra642

342 ra463

343 ra467

344 ra529

345 ra468

346 ra140

347 ra141

348 no22Y

349 ra591

350 ra638

351 ra650

352 ra592

353 ra578

354 ra604

355 ra373

356 ra171

357 ra110

358 ra107

359 ra93

360 ra370

361 ra92

362 ra79

363 ra639

364 ra649

365 ra546

366 ra554

367 mW

368 ra324

369 ra327

370 ra605

371 Ra385

372 ra354

373 ra58

374 ra314

375 ra486

376 ra567

377 napA

378 ra566

379 ra148

380 ra167 & ra78

381 ra71

382 ra334 & ra487

383 ra333

384 ra452

385 ra306

386 ra637

387 ra587

388 ra586

389 ra643

390 ra453

391 ra308

392 ra305

393 ra661

394 ra647

395 ra326

396 ra323

397 ra342

398 ra496

399 ra332

400 ra593

401 ra81

402 ra663

403 ra640

404 ra646

405 ra636

406 ra652

407 ra515

408 ra520

409 ra94

410 ra137

411 ra495 & ra531

412 ra641

413 ra651

414 ra612

415 ra500

416 ra644

417 ra399

418 ra98

419 ra645

420 Pyl

421 DPyl

422 ra662

423 ra653

424 ra491

425 ra577

426 ra70

427 ra95

428 ra97

429 ra136

430 ra96

431 ra514

432 ra654

433 ra657

434 ra511

435 ra366

436 pnaC

437 ra615

438 pnaT

439 ra624

440 ra526

441 ra525

442 ra471

443 ra613

444 ra599

445 ra553

446 ra626

447 ra633

448 ra628

449 ra60

450 ra73

451 ra175

452 ra606

453 ra398

454 ra494

455 ra501

456 ra503

457 ra611

458 ra353

459 ra616

460 ra629

461 ra504

462 pnaA

463 ra318

464 ra614

465 ra630

466 ra512

467 ra319

468 Pqa

469 ra619

470 ra627

471 ra623

472 ra358

473 ra346

474 ra492

475 ra493

476 ra617

477 ra622

478 ra502

479 ra655

480 ra618

481 ra625

482 ra621

483 ra631

484 pnaG

485 ra607

486 ra656

487 ra620

488 ra668

489 ra635

490 ra472

491 ra569

492 ra632

493 ra634

494 ra570

495 ra595

496 ra311

497 ra304

498 ra303

499 ra571

500 ra309

501 ra402

502 ra322

503 ra349

504 ra408

505 ra572

506 ra580

507 Ala

508 mAla

509 dAla

510 ChA

511 Pro

512 Val

513 mVal

514 Leu

515 dLeu

516 mLeu

517 mdLeu

518 mLeu

519 HoSerMe

520 Phe

521 Nal

522 Nva

523 PhF

524 PhG

525 dPhe

526 mG

527 mNle

528 mPhe

529 mSerBu

530 mdPhe

531 mIle

The monomers RbAsp, dD, D, and SbAsp have more than one hydroxyl groups. In some embodiments, the hydroxyl group that serves as a linkage point to the adjacent residues in each of these monomers is illustrated in Scheme 2 above. In some embodiments, the other hydroxyl group in these monomers can be used as a linkage point to the adjacent residues.

In some embodiments, disclosed herein is a compound of Formula VIII or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, R can be

R¹, R², R³, R⁴, and R⁵ can be each independently selected from hydrogen, hydroxyl, alkoxy, cyano, alkylthio, amino, and alkylamino, and

wherein

can be a resin; wherein one, two, three, or four of A¹, A², A³, A⁴, and A⁵ can be N or P with the remaining being CH; wherein one, two, three, or four of B¹, B², B³ and B⁴ can be O, N, or S with the remaining being CH or CH₂ as appropriate; wherein

can be a single or double bond.

In some embodiments, X₁ can be O or NR⁶; Y can be —C(O)— or

X₂ can be (CH₂)_(m), O, OC(O), NR⁶, NR⁶C(O); Z can be

W can be O, CH, CH₂, CR⁹, or C R¹⁰R¹¹; can be L₁ and L₂ can be each independently a direct bond, substituted or unsubstituted —(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted —(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted —(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted —(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)S(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)S(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-C(O)—, —O—, —NH—, —S—, —S(O)—, —SO₂—, —Si—, and —B—, wherein each alkyl, alkenyl, and alkynyl group may be optionally substituted with alkyl, alkoxy, amino, hydroxyl, sulfhydryl, halogen, carboxyl, oxo, cyano, nitro, or trifluoromethyl.

L₃ can be a direct bond, substituted or unsubstituted —(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted —(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted —(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted —(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)S(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-NR¹⁸—, substituted or unsubstituted —(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-NR¹⁸—, substituted or unsubstituted —(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-NR¹⁸—, substituted or unsubstituted —(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)S(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-C(O)—, substituted or unsubstituted —(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-C(O)—, substituted or unsubstituted —(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)OC(O)(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)NH(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkynyl-C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-C(O)—, wherein each alkyl, alkenyl and alkynyl group may be optionally substituted with alkyl, alkoxy, amino, hydroxyl, sulfhydryl, halogen, carboxyl, oxo, cyano, nitro, or trifluoromethyl.

Each m can be independently an integer selected from 0, 1, 2, 3, 4, 5, and 6; each n is independently an integer selected from 0, 1, 2, 3, 4, 5, and 6; R⁶ is hydrogen or alkyl; R⁷ and R⁸ are each independently selected from hydrogen, hydroxy, alkyl, alkoxy, cyano, alkylthio, amino, and alkylamino, and OPG, wherein OPG is a protecting group; R⁹, R¹⁰, and R¹¹ are each independently selected from hydrogen, hydroxy, alkyl, alkoxy, cyano, alkylthio, amino, and alkylamino, and OPG, wherein OPG is a protecting group.

The Effector Domain can have Formula (A):

R¹², R¹⁴, R¹⁶, and R¹⁸ can be each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅. R¹³, R¹⁵, and R¹⁷ are each independently the sidechains of naturally occurring amino acids and their modified forms including but are not limited to D-amino acid configuration, or hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, substituted or unsubstituted (CH₂)_(n)-aryl, substituted or unsubstituted (CH₂)_(n)-heteroaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹, (CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹, (CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹, (CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²⁰R²¹, (CH₂)_(n)SR¹⁹, (CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)R¹⁹, or —(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹.

R¹² and R¹³, R¹⁴ and R¹⁵, R¹⁶ and R¹⁷ can be covalently connected to form a substituted or unsubstituted 5-, 6-, or 7-membered heterocycle. Each k can be independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Each j can be independently an integer selected from 0, 1, and 2. R¹⁹, R²⁰, R²¹, and R²² can be each independently hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, or heteroalkylaryl.

Or R¹⁹ and R²² are as described above, and R²⁰ and R²¹, together with the N atom to which they are attached, form a substituted or unsubstituted 5-, 6-, or 7-membered heterocycloalkyl or a substituted or unsubstituted 5-membered heteroaryl, wherein each of the above groups listed for R¹³, R¹⁵, and R¹⁷ may be optionally independently substituted with 1 to 3 groups selected from halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹, (CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹, (CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹, (CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²⁰R²¹, (CH₂)_(n)SR¹⁹, (CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)R¹⁹, or —(CH₂)_(n)NR²²S(O)NR²⁰R²¹.

Or the Effector Domain can have Formula (B):

Each k can be independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R²³ can be a hydrogen or alkyl; X₃ can be substituted or unsubstituted —(C₁-C₃₀)alkyl-, alkenyl-, alkynyl-with each carbon individually assuming one of the following redox states: CH₂, CH—OH, C(O);

Or the Effector Domain can have Formula (C):

X₄ can be substituted or unsubstituted —(C₁-C₃₀)alkyl-, alkenyl-, alkynyl-with each carbon individually assuming one of the following redox states: CH₂, CH—OH, C(O).

Or the Effector Domain has Formula (D):

R²⁴ and R²⁵ are each a hydrogen or alkyl; X₅ can be substituted or unsubstituted —(C₁-C₃₀)alkyl-, alkenyl-, alkynyl-with each carbon individually assuming one of the following redox states: CH₂, CH—OH, C(O).

Or the Effector Domain can be Formula (E):

X₆ can be substituted or unsubstituted —(C₁-C₃₀)alkyl-, alkenyl-, alkynyl-with each carbon individually assuming one of the following redox states: CH₂, CH—OH, C(O).

In some embodiments, L₃ is not with R²⁶ being hydrogen or alkyl.

In some embodiments, R is not, wherein R³ is hydrogen, hydroxyl, or OPG, wherein PG is a protecting group, or

wherein

is a resin; wherein R² is hydrogen, hydroxyl, or alkoxy; and wherein R¹, R⁴, and R⁵ are each independently hydrogen or no substituent as dictated by chemical bonding; wherein

is a single or double bond.

In some embodiments, L₁ and L₂ not each independently direct bond, substituted or unsubstituted —(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)—, substituted or unsubstituted —(CH₂)_(n)C(O)(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₁-C₆)alkyl-, substituted or unsubstituted —(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkenyl-, substituted or unsubstituted —(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)O(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)O(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)NH(C₁-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)NH(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)S(C₂-C₆)alkynyl-, substituted or unsubstituted —(CH₂)_(n)C(O)(CH₂)_(n)S(C₂-C₆)alkynyl-, wherein each alkyl, alkenyl, and alkynyl group may be optionally substituted with alkyl, alkoxy, amino, carboxyl, cyano, nitro, or trifluoromethyl.

In some embodiments, the Effector Domain is a compound of Formula (F)

R¹², R¹⁴, R^(14′), R¹⁶, and R²⁷ are not each independently hydrogen or alkyl and R¹³, R¹⁴, R^(14′), and R¹⁶ are not each independently hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹, (CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹, (CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹, (CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²⁰R²¹, (CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)_(j)R¹⁹, or —(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹; n is an integer selected from 0, 1, 2, 3, 4, 5, and 6; j is an integer selected from 0, 1, and 2.

R¹⁹, R²⁰, R²¹, and R²² are each independently hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, or heteroalkylaryl, or R¹⁹ and R²² are as described above, and R²⁰ and R²¹, together with the N atom to which they are attached, form a substituted or unsubstituted 5-, 6-, or 7-membered heterocycloalkyl or a substituted or unsubstituted 5-membered heteroaryl.

Each of the above groups listed for R¹³, R¹⁵, and R¹⁷ may be optionally independently substituted with 1 to 3 groups selected from halogen, amino, cyano, nitro, trifluoromethyl, alkyl, alkenyl, alkynyl, cycloalkyl, perfluoroalkyl, alkoxy, alkylamino, alkylthio, aryl, alkylaryl, heteroalkyl, heterocycloalkyl, heteroaryl, heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹, (CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹, (CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹, (CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²⁰R²¹, (CH₂)_(n)SR¹⁹, (CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)_(j)R¹⁹, or —(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹.

In some embodiments, L₃ in Formula (VII) is —CH₂CH₂—, R is

R¹, R⁴, R⁵ and R⁶ are each hydrogen; R² and R³ are each methoxy; m=0; Y is I;

X₂ is O or NR⁶C(O); Li is —CH₂—C(O)— or —(CH₂)₂C(O)—; Z is

L₂ is —OCO—CH═CH—(CH₂)₂N(Me)-. In some embodiments, X₂ is O and Li is —CH₂—C(O)—. In some embodiments, X₂ is NR⁶C(O) and Li is —(CH₂)₂C(O)—.

In some embodiments, the effector domain can be Formula (G)

Wherein R¹², R¹⁴, R^(14′), and R¹⁶ are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅.

R¹³, R¹⁵, R^(15′) and R¹⁷ are each independently the sidechains of naturally occurring amino acids and their modified forms including but are not limited to D-amino acid configuration, or hydrogen, halogen, amino, cyano, nitro, trifluoromethyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted perfluoroalkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted alkylamino, substituted or unsubstituted alkylthio, substituted or unsubstituted aryl, substituted or unsubstituted alkylaryl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroalkylaryl, substituted or unsubstituted (CH₂)_(n)-aryl, substituted or unsubstituted (CH₂)_(n)-heteroaryl, (CH₂)_(n)CN, (CH₂)_(n)CF₃, (CH₂)_(n)C₂F₅, (CH₂)_(n)OR¹⁹, (CH₂)_(n)C(O)R¹⁹, (CH₂)_(n)C(O)OR¹⁹, (CH₂)_(n)OC(O)R¹⁹, (CH₂)_(n)NR²⁰R²¹, (CH₂)_(n)C(O)NR²⁰R²¹, (CH₂)_(n)NR²²C(O)R¹⁹, (CH₂)_(n)NR²²C(O)OR¹⁹, (CH₂)_(n)NR²²C(O)NR²⁰R²¹, (CH₂)_(n)SR¹⁹, (CH₂)_(n)S(O)_(j)NR²⁰R²¹, (CH₂)_(n)NR²²S(O)_(j)R¹⁹, or —(CH₂)_(n)NR²²S(O)_(j)NR²⁰R²¹. R¹² and R¹³, R¹⁴ and R¹⁵, R^(14′) and R^(15′), R¹⁶ and R¹⁷ can be covalently connected to form a substituted or unsubstituted 5-, 6-, or 7-membered heterocycle.

In some embodiments, disclosed herein is a method of using a hybrid cyclic library based on the immunophilin ligand family of natural products FK506 and rapamycin, to screen for compounds for treating cancer. In some embodiments, disclosed herein is a method of using a hybrid cyclic library based on the immunophilin ligand family of natural products FK506 and rapamycin, to screen for compounds for treating autoimmune disease.

In some embodiments, the Rapafucin compounds in the present disclosure can have a structure according to Formula (IX) or Formula (X) or an optically pure stereoisomer or pharmaceutically acceptable salt thereof.

The amino acid moieties with R₁, R₂, R₃, and R₄ can be selected from Table 2 below illustrating the amino acid monomers used for the present disclosure. In some embodiments, the amino acid moieties with R₁ and R₃ can be selected from the group consisting of

or R₁ or R₃ together with nitrogen to form

In some embodiments, the amino acid moieties with R₂ and R₄ can be selected from the group consisting of

The macrocyclic natural products FK506 and rapamycin are approved immunosuppressive drugs with important biological activities. Both have been shown to inhibit T-cell activation, each with distinct mechanisms. In addition, rapamycin has been shown to have strong anti-proliferative activity. FK506 and rapamycin share an extraordinary mode of action; they act by recruiting an abundant and ubiquitously expressed cellular protein, the prolyl cis-trans isomerase FKBP, and the binary complexes subsequently bind to and allosterically inhibit their target proteins calcineurin and mTOR, respectively. Structurally, FK506 and rapamycin share a similar FKBP-binding domain but differ in their effector domains. In FK506 and rapamycin, nature has taught us that switching the effector domain of FK506 to that in rapamycin, it is possible to change the targets from calcineurin to mTOR. The generation of a rapafucin library of macrocyles that contain FK506 and rapamycin binding domains should have great potential as new leads for developing drugs to be used for treating diseases.

A variety of methods exist for the generation of compound libraries for developing and screening potentially useful compounds in treating diseases. One such method is the development of encoded libraries, and particularly libraries in which each compound includes an amplifiable tag. Such libraries include DNA-encoded libraries in which a DNA tag identifying a library member can be amplified using molecular biology techniques, such as the polymerase chain reaction (PCR). The use of such methods for producing libraries of rapafucin macrocyles that contain FK506-like and rapamycin-like binding domains has yet to be demonstrated. Thus, there remains a need for DNA-encoded rapafucin libraries of macrocyles that contain FK506-like and rapamycin-like binding domains.

In one aspect, provided herein is a tagged macrocyclic compound that comprises: an FK506 binding protein binding domain (FKBD); an effector domain; a first linking region; and a second linking region; wherein the FKBD, the effector domain, the first linking region, and the second linking region together form a macrocycle; and wherein at least one of the FKBD, the effector domain, the first linker, and the second linker can be operatively linked to one or more oligonucleotides (D) which can identify the structure of at least one of the FKBD, the effector domain, the first linker, and the second linker.

In certain embodiments, provided herein is a tagged macrocyclic compound of Formula (XI):

In some embodiments, h, i, j, and k are each independently an integer from 0-20, provided that at least one of h, i, j, and k is not 0; and D is an oligonucleotide that can identify at least one of the FKBD, the Effector Domain, the Linking Region A, or the Linking Region Z, where the solid lines linking the FKBD, the Effector Domain, the Linking Region A, and/or the Linking Region Z indicate an operative linkage and the squiggle lines indicate an operative linkage. In certain embodiments, oligonucleotide (D) can be operatively linked to at least one of the FKBD, the Effector Domain, the Linking Region A, or the Linking Region Z.

In some embodiments, provided herein is a tagged macrocyclic compound of Formula (XII) or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof:

In some embodiments, Ring A is a 5-10 membered aryl, cycloalkyl, heteroaryl or heterocycloalkyl, optionally substituted with 1-17 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin; J is independently at each occurrence selected from the group consisting of —C(O)NR⁶—.

wherein R⁶ is each hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R′ is hydrogen, alkyl, arylalkyl, or haloalkyl; D is independently at each occurrence an oligonucleotide; L^(b) and L^(c) are independently at each occurrence selected from the group consisting of bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)OC(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)—S—C₁₋₆ alkylene (CH₂)_(n)—, and optionally substituted (CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or 2 groups independently selected from the group consisting of of halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl, arylalkyl,

or and

wherein R^(N) is aryl, alkyl, or arylalkyl; X is O, S or NR⁸, wherein R⁸ is hydrogen, hydroxy, OR⁹, NR¹⁰R¹¹ alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ are each independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionally substituted with 1-10 substituents, each of which is selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, amino, alkylamino, dialkylamino, arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen, hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is each independently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, arylalkyl, or heteroaryl; Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸, CR¹⁸, N, or and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl;

L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond, —O—, —NR¹⁹—, —SO—, —SO₂—, (CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6 membered heteroaryl, optionally substituted with 1-4 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxyl, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino and

wherein each R¹⁹, R²⁰, and R²¹ is independently is selected from the group consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴ are each independently hydrogen or alkyl;

n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula (XIIa):

In some embodiments, each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i) is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ is independently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ is hydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸ are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is independently selected from the group consisting of hydrogen, halo, amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnatural amino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

or wherein the Effector Domain has Formula (XIIc):

wherein each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R²⁹ is a hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³² are each independently hydrogen or alkyl; X³ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIId):

wherein X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIe):

wherein R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; provided that when R is

L is ethylene, X is O, W is

V is

Z is

-L⁶-L⁷-L⁸-is

then -L¹-L²-L³-L⁴-L⁵-is not

wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring C substituted with at least one

or wherein at least one of the linking groups selected from Table 1 is substituted with at least one

In another aspect, provided herein is a compound library that comprises a plurality of distinct tagged macrocyclic compounds according to any of the above. In certain embodiments, provided herein is a compound library that comprises at least about 10² distinct tagged macrocyclic compounds according to any of the above. In certain embodiments, provided herein is a compound library that comprises from about 10² to about 10¹⁰ distinct tagged macrocyclic compounds according to any of the above.

In a further aspect, provided herein is a method of making a library of tagged macrocyclic compounds as disclosed herein, the method comprising synthesizing a plurality of distinct tagged macrocyclic compounds according to any of the above.

In a still further aspect, provided herein is a method of making a tagged macrocyclic compound as disclosed herein, the method comprising operatively linking at least one oligonucleotide (D) to at least one of an FKBD, an effector domain, a first linking region, and a second linking region, and forming a macrocyclic ring comprising the FKBD, the effector domain, the first linking region, and the second linking region.

In certain embodiments, provided herein is a method of making a tagged macrocyclic compound as disclosed herein, the method comprising macrocyclic compound to at least one oligonucleotide (D), the macrocyclic compound comprising an FKBD, an effector domain, a first linking region, and a second linking region, wherein the FKBD, the effector domain, the first linking region, and the second linking region together form a macrocycle; and wherein the at least one oligonucleotide (D) can identify the structure of at least one of the FKBD, the effector domain, the first linking region, and the second linking region.

In yet a further aspect, the method of making a tagged macrocyclic compound comprises: operatively linking a compound of Formula (XIII):

to a compound of Formula (XIV):

Q′-L^(c)-D   Formula (XIV)

In some embodiments, L and L′ are independently at each occurrence: a bond, —O—, —NR¹⁹—, —SO—, —SO₂—, —(CH₂)_(n)—,

or a linking group selected from Table 1 wherein Ring C is a 5-6 membered heteroaryl, optionally substituted with 1-4 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino; wherein R¹⁹ is selected from the group consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴ are each independently hydrogen or alkyl; Q and Q′ are each independently selected from the group consisting of N₃, —C≡CH, NR⁶R⁷, —COOH, —ONH₂, —SH, —NH₂,

(C═O)R′,

wherein R⁶ and R⁷ is each independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; and R′ is hydrogen, alkyl, arylalkyl, or haloalkyl; L^(b) and L^(c) are independently at each occurrence selected from the group consisting of a bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)OC(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)—S—C₁₋₆ alkylene-(CH₂)_(n)—, and optionally substituted (CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or 2 groups independently selected from the group consisting of of halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl;

D is an oligonucleotide; h, i, j, and k are each independently an integer from 0-20, provided that at least one of h, i, j, and k is not 0; n is an integer from 1-5; m is an integer from 1-5.

In another aspect, provided herein is a method of making a tagged macrocyclic compound, the method comprising operatively linking a compound of Formula (XII):

with a compound of Formula (XIV):

Q′-L^(c)-D   Formula (XIV)

Ring A is a 5-10 membered aryl, cycloalkyl, heteroaryl or heterocycloalkyl, optionally substituted with 1-17 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin;

L^(b) and L^(c) are independently selected from the group consisting of a bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)OC(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)—S—C₁₋₆ alkylene-(CH₂)_(n)—, and optionally substituted (CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or 2 groups independently selected from the group consisting of of halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl;

Q and Q′ are independently selected from the group consisting of —N₃, —C≡CH, NR⁶R⁷, —COOH, —ONH₂, —SH, —NH₂,

(C═O)R′,

wherein R⁶ and R⁷ is each independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; and R′ is hydrogen, alkyl, arylalkyl, or haloalkyl; X is O, S or NR⁸, wherein R⁸ is hydrogen, hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ are each independently hydrogen or alkyl; V¹ and V² are each independently

wherein Ring B is a 4-10 membered heterocycloalkyl, optionally substituted with 1-10 substituents, each of which is selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino, arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen, hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is each independently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, arylalkyl, or heteroaryl;

Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸, CR¹⁸, N, and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl;

L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond, —O—, —NR¹⁹—, —SO—, —SO₂—, —(CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6 membered heteroaryl, optionally substituted with 1-4 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino and

wherein R¹⁹ is selected from the group consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴ are each independently hydrogen or alkyl;

n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula (XIIa):

each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i) is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ is independently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ is hydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸ are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is independently selected from the group consisting of hydrogen, halo, amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnatural amino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10;

or wherein the Effector Domain has Formula (XIIc):

each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R²⁹ is hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³² are each independently hydrogen or alkyl; X³ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIId):

X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIe):

R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2;

or wherein the Effector Domain has Formula (XIIf):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, on

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; and provided that when Ring A is

L^(a) is ethylene, X is O, W is

V¹ is

V² is

Z is

-L⁶-L⁷-L⁸-is

and -L¹-L²-L³-L⁴-L⁵-is not

D is an oligonucleotide; wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring C substituted with at least one

or wherein at least one of the linking groups selected from Table 1 is substituted with at least one

In yet another aspect, provided herein is a method for identifying one or more compounds that bind to a biological target the method comprising: (a) incubating the biological target with at least a portion of the plurality of distinct tagged macrocyclic compounds of the compound library of claim 2 to make at least one bound compound and at least one unbound compound of the plurality of distinct tagged macrocyclic compounds; (b) removing the at least one unbound compound; and (c) sequencing each of the oligonucleotides (D) of the at least one bound compound.

In certain embodiments, the DNA-encoded library can be a single pharmacophore library, wherein only one chemical moiety can be attached to a single strand of DNA, as described in, e.g., Neri & Lerner, Annu. Rev. Biochem. (2018) 87:5.1-5.24, which is hereby incorporated by reference in its entirety. In certain embodiments, the DNA-encoded library can be a dual pharmacophore library, wherein two independent molecules can be attached to the double strands of DNA, as described in, e.g., Id; Mannocci et al., Chem. Commun. (2011) 47:12747-53, which is hereby incorporated by reference in its entirety.

In a further aspect, provided herein is a method of making a library of tagged macrocyclic compounds, the method comprising synthesizing a plurality of distinct tagged macrocyclic compounds. In certain embodiments, each tagged macrocyclic compound of the plurality of distinct tagged macrocyclic compounds comprising a macrocyclic compound operatively linked to at least one oligonucleotide (D). In certain embodiments, each compound of the plurality of distinct tagged macrocyclic compounds of the compound library comprises a macrocyclic compound operatively linked to at least one oligonucleotide (D). In certain embodiments, the macrocyclic compound comprising an FKBD, an effector domain, a first linking region, and a second linking region. In certain embodiments, the FKBD, the effector domain, the first linking region, and the second linking region together form a macrocycle. In certain embodiments, each of the at least one oligonucleotide (D) can identify at least one of the FKBD, the effector domain, the first linking region, and the second linking region of each of the plurality of distinct tagged macrocyclic compounds. In certain embodiments, each compound of the plurality of distinct tagged macrocyclic compounds of the compound library comprises a compound of Formula (A) (as above-defined). In certain embodiments, each compound of the plurality of distinct tagged macrocyclic compounds of the compound library comprises a compound of Formula (I) (as above-defined herein). In certain embodiments, each compound of the plurality of distinct tagged macrocyclic compounds of the compound library can be a reaction product of operatively linking a compound of Formula (B) (as above-defined herein) with a compound of Formula (C) (as above-defined herein). In certain embodiments, each compound of the plurality of distinct tagged macrocyclic compounds of the compound library can be a reaction product of operatively linking a compound of Formula (B′) (as above-defined herein) with a compound of Formula (C) (as above-defined herein).

In certain embodiments, the method of synthesizing a library of compounds can be selected from the group consisting of the split-and-pool method, DNA-templated library synthesis (DTS), encoded self-assembling chemical (ESAC) library synthesis, DNA-recorded library synthesis, DNA-directed library synthesis, DNA-routing, and 3-D proximity-based library synthesis (YoctoReactor). As a person of ordinary skill in the art would be aware, various techniques for synthesizing the library of tagged macrocyclic compounds are described in, e.g., Neri & Lerner, Annu. Rev. Biochem. (2018) 87:5.1-5.24; Roman et al., SLAS Discov. (2018) 23(5):387-396; Lim, C&EN, (2017) 95 (29):10-10; Halford, C&EN, (2017) 95(25): 28-33; Estevez, Tetrahedron: Asymmetry. (2017) 28:837-842; Neri, Chembiochem. (2017) 4; 18(9):827-828; Yuen & Franzini, Chembiochem. (2017) 4; 18(9):829-836; Skopic et al., Chem Sci. (2017) 1; 8(5):3356-3361; Shi et al.; Bioorg Med Chem Lett. (2017) 1; 27(3):361-69; Zimmermann & Neri, Drug Discov Today. (2016) 21(11):1828-1834; Satz et al., Bioconjug Chem. (2015) 19; 26(8):1623-32; Ding et al., ACS Comb Sci. (2016) 10; 18(10):625-629; Arico-Muendel, MedChemComm, (2016) 7(10): 1898-1909; Skopic, MedChemComm, (2016) 7(10): 1957-1965; Satz, CS Comb. Sci. (2016) 18 (7):415-424; Tian et al., MedChemComm, (2016) 7(7): 1316-1322; Salamon et al., ACS Chem Biol. (2016) 19; 11(2):296-307; Satz et al., Bioconjug Chem. (2015) 19; 26(8):1623-32; Connors et al., Curr Opin Chem Biol. (2015) 26:42-7; Blakskjaer et al., Curr Opin Chem Biol. (2015) 26:62-71; Scheuermann & Neri, Curr Opin Chem Biol. (2015) 26:99-103; Franzini et al., Angew Chem Int Ed Engl. (2015) 23; 54(13):3927-31; Franzini et al., Bioconjug Chem. (2014) 20; 25(8):1453-61; Franzini, Neri & Scheuermann, Acc Chem Res. (2014) 15; 47(4):1247-55; Mannocci et al., Chem. Commun. (2011) 47:12747-53; Kleiner et al., Chem Soc Rev. (2011) 40(12): 5707-17; Clark, Curr Opin Chem Biol. (2010) 14(3):396-403; Mannocci et al., Proc Natl Acad Sci USA. (2008) 18; 105(46):17670-75; Buller et al., Bioorg Med Chem Lett. (2008) 18(22):5926-31; Scheuermann et al., Bioconjugate Chem. (2008) 19:778-85; Zimmerman et al., ChemBioChem (2017) 18(9):853-57, and Cuozzo et al., ChemBioChem (2017), 18(9):864-71, each of which is hereby incorporated by reference in its entirety.

In some embodiments, the method of synthesizing a library of tagged macrocyclic compounds comprises DNA-recorded library synthesis, in which encoding and library synthesis take place separately, as described in, e.g. Shi et al., Bioorg Med Chem Lett. (2017) 1; 27(3):361-369; Kleiner et al., Chem Soc Rev. (2011) 40(12): 5707-17. In certain embodiments, the DNA-recorded library synthesis c comprises split-and-pool methods, which are described in, e.g., Krall, Scheuermann & Neri, Angew Chem. Int. Ed Engl. (2013) 28; 52(5):1384-402; Mannocci et al., Chem. Commun. (2011) 47:12747-53; and U.S. Pat. No. 7,989,395 to Morgan et al., each of which is hereby incorporated by reference in its entirety. In certain embodiments, the split-and-pool method comprises successive chemical ligation of oligonucleotide tags to an initial oligonucleotide (or headpiece), which can be covalently linked to a chemically generated entity by successive split-and-pool steps. In certain embodiments, during each split step, a chemical synthesis step can be performed along with an oligonucleotide ligation step.

In some embodiments, the library can be synthesized by a sequence of split-and-pool cycles, wherein an initial oligonucleotide (or headpiece) can be reacted with a first set of building blocks (e.g., a plurality of FKBD building blocks). For each building block of the first set of building blocks (e.g., each FKBD building block), an oligonucleotide (D) can be appended to the initial oligonucleotide (or headpiece) and the resulting product can be pooled (or mixed), and subsequently split into separate reactions. Subsequently, in certain embodiments, a second set of building blocks (e.g., a plurality of effector domain building blocks) can be added, and an oligonucleotide (D) can be appended to each building block of the second set of building blocks. In certain embodiments, each oligonucleotide (D) identifies a distinct building block.

In some embodiments, the method of synthesizing a library of tagged macrocyclic compounds comprises DNA-directed library synthesis, in which DNA both encodes and templates library synthesis as described in, e.g. Kleiner et al., Bioconjugate Chem. (2010) 21, 1836-41; and Shi et. al, Bioorg Med Chem Lett. (2017) 1; 27(3):361-369, each of which is hereby incorporated by reference in its entirety. In certain embodiments, the DNA-directed library synthesis comprises the DNA-templated synthesis (DTS) method as described in, e.g., Mannocci et al., Chem. Commun. (2011) 47:12747-53, Franzini, Neri & Scheuermann, Acc Chem Res. (2014) 15; 47(4):1247-55; and Mannocci et al., Chem. Commun. (2011) 47:12747-53, each of which are hereby incorporated by reference in its entirety. In certain embodiments, the DTS method comprises DNA oligonucleotides that not only encode but also direct the construction of the library. See Buller et al., Bioconjugate Chem. (2010) 21, 1571-80, which is hereby incorporated by reference in its entirety. In certain embodiments different building blocks can be incorporated into molecules using DNA-linked reagents that can be forced into proximity by base pairing between their DNA tags. See Gartner et al., Science (2004) 305:1601-05, which is hereby incorporated by reference in its entirety. In certain embodiments, a library of long oligonucleotides can be synthesized first as a template for the DNA-encoded library. In certain embodiments, the oligonucleotides can be subjected to sequence-specific chemical reactions through immobilization on resin tagged with complementary DNA sequences. See Wrenn & Harbury, Annu. Rev. Biochem. (2007) 76:331-49, which is hereby incorporated by reference in its entirety.

In certain embodiments, the DNA-directed library synthesis comprises 3-D proximity-based library synthesis, also known as YoctoReactor technology, which is described in, e.g., Blakskjaer et al., Curr Opin Chem Biol. (2015) 26:62-7, which is hereby incorporated by reference in its entirety.

In certain embodiments, the method of synthesizing a library of tagged macrocyclic compounds comprises encoded self-assembling chemical (ESAC) library synthesis, also known as double-pharmacophore DNA-encoded chemical libraries, as described in, e.g., Mannocci et al., Chem. Commun. (2011) 47:12747-53; Melkko et al., Nat. Biotechnol. (2004) 22(5):568-74; Scheuermann et al., Bioconjugate Chem. (2008) 19:778-85; and U.S. Pat. No. 8,642,215 to Neri et al. each of which is hereby incorporated by reference in its entirety. In certain embodiments, synthesizing a library of tagged macrocyclic compounds by ESAC synthesis comprises, for example, non-covalent combinatorial assembly of complementary oligonucleotide sub-libraries, in which each sub-library can include a first oligonucleotide appended to a first building block, wherein the first oligonucleotide comprises a coding domain that identifies the first building block, and a hybridization domain, which self-assembles to a second oligonucleotide appended to a second building block, second oligonucleotide comprising a coding domain that identifies the second building block, and a hybridization domain that self-assembles to the first oligonucleotide.

In some embodiments, the method of synthesizing a library of tagged macrocyclic compounds comprises DNA-routing, as described in, e.g. Clark, Curr Opin Chem Biol. (2010) 14(3):396-403, which is hereby incorporated by reference in its entirety.

In certain embodiments, oligonucleotide ligation can utilize one of several methods that would be appreciated be a person of ordinary skill in the art, described, for example, in Zimmermann & Neri, Drug Discov. Today. (2016) 21(11):1828-1834; and Keefe et al., Curr Opin Chem Biol. (2015) 26:80-88, each of which are hereby incorporated by reference in its entirety. In certain embodiments, the oligonucleotide ligation can be an enzymatic ligation. In certain embodiments, the oligonucleotide ligation can be a chemical ligation.

In certain embodiments, the ligation comprises base-pairing a short, complementary “adapter” oligonucleotide to single-stranded oligonucleotides to either end of the ligation site, allowing ligation of single-stranded DNA tags in each cycle. See Clark et al., Nat. Chem. Biol. (2009) 5:647-54, which is hereby incorporated by reference in its entirety. In certain embodiments, the oligonucleotide ligation comprises utilizing 2-base overhangs at the 3′ end of the headpiece and of each building block's DNA tag to form sticky ends for ligation. In certain embodiments, the sequences of the overhangs can depend on the cycle but not on the building block, so that any DNA tag can be ligated to any DNA tag from the previous cycle, but not to a truncated sequence. See id. In certain embodiments, the oligonucleotide ligation step can utilize oligonucleotides of opposite sense for subsequent cycles, with a small region of overlap in which the two oligonucleotides are complementary. In certain embodiments, in lieu of ligation, DNA polymerase can be used to fill in the rest of the complementary sequences, creating a double-strand oligonucleotide comprising both tags. In certain embodiments, the oligonucleotide ligation can be chemical. While not wishing to be bound by theory, it is thought that chemical ligation may permit greater flexibility with regard to solution conditions and may reduce the buffer exchange steps necessary. See Keefe et al., Curr Opin Chem Biol. (2015) 26:80-88, which is hereby incorporated by reference in its entirety.

In certain embodiments, provided herein is a method for identifying one or more compounds that bind to a biological target, the method comprising: (a) incubating the biological target with at least a portion of a plurality of distinct tagged macrocyclic compounds of a compound library to make at least one bound compound and at least one unbound compound of the plurality of distinct tagged macrocyclic compounds; (b) removing the at least one unbound compound; (c) sequencing each of the at least one oligonucleotide (D) of the at least one bound compound. In certain embodiments, each compound of the plurality of distinct tagged macrocyclic compounds of the compound library comprises a macrocyclic compound operatively linked to at least one oligonucleotide (D). In certain embodiments, the macrocyclic compound comprises an FKBD, an effector domain, a first linking region, and a second linking region. In certain embodiments, the FKBD, the effector domain, the first linking region, and the second linking region together form a macrocycle. In certain embodiments, each at least one oligonucleotide (D) can identify at least one of the FKBD, the effector domain, the first linking region, and the second linking region of each of the plurality of distinct tagged macrocyclic compounds. In certain embodiments, each compound of the plurality of distinct tagged macrocyclic compounds of the compound library comprises a compound of Formula (A) (as above-defined). In certain embodiments, each compound of the plurality of distinct tagged macrocyclic compounds of the compound library comprises a compound of Formula (I) (as above-defined). As a person of ordinary skill in the art would be aware, various techniques for synthesizing the library of tagged macrocyclic compounds are described in, e.g., Kuai et al., SLAS Discov. (2018) 23(5):405-416; Brown et al., Annu. Rev. Biochem. (2018) 87:5.1-5.24; Roman et al., SLAS Discov. (2018) 23(5):387-396; Amigo et al., SLAS Discov. (2018) 23(5):397-404; Shi et al., Bioconjug Chem. (2017) 20; 28(9):2293-2301; Machutta et al., Nat Commun. (2017) 8:16081; Li et al., Chembiochem. (2017) 4; 18(9):848-852; Satz et al., ACS Comb Sci. (2017) 10; 19(4):234-238; Denton & Krusemark, MedChemComm, (2016) 7(10): 2020-2027; Eidam & Satz, MedChemComm, (2016) 7(7): 1323-1331; Bao et al., Anal. Chem., (2016) 88 (10):5498-5506; Decurtins et al., Nat Protoc. (2016) 11(4):764-80; Harris et al., J. Med. Chem. (2016) 59 (5):2163-78; Satz, ACS Chem Biol. (2016) 16; 10(10):2237-45; Chan et al., Curr Opin Chem Biol. (2015) 26:55-61; Franzini et al., Chem Commun. (2015) 11; 51(38):8014-16; and Buller et al., Bioorg Med Chem Lett. (2010) 15; 20(14):4188-92. each of which is hereby incorporated by reference in its entirety.

In certain embodiments, the incubating step can be performed under conditions suitable for at least one of the plurality of distinct tagged macrocyclic compounds of the compound library to bind to the biological target. A person of ordinary skill in the art would understand what conditions would be considered suitable for at least one of the plurality of distinct tagged macrocyclic compounds of the compound library to bind to the biological target.

In certain embodiments, the identifying one or more compounds that bind to a biological target comprises a bind-wash-elute procedure for molecule selection as described in, e.g., Ding et al., ACS Med. Chem. Lett. (2015) 7; 6(8):888-93, which is hereby incorporated by reference in its entirety. In certain embodiments, the incubating step (a comprises contacting the plurality of tagged compounds in the compound library with a target protein, wherein the target protein can be immobilized on a substrate (e.g., resin). In certain embodiments, the removing step (b) comprises washing the substrate to remove the at least one unbound compound. In certain embodiments, the sequencing step (c) comprises sequencing the at least one oligonucleotide (D) to identify which of the plurality of tagged compounds bound to the target protein.

In certain embodiments, the identifying one or more compounds that bind to a biological target comprises utilizing unmodified, non-immobilized target protein. Such methods, which can utilize a a ligate-crosslink-purify strategy are described in, e.g., Shi et al., Bioconjug. Chem. (2017) 20; 28(9):2293-2301, which is hereby incorporated by reference in its entirety. In certain embodiments, other methods for identifying the one or more compounds that bind to the biological target can be utilized. Such methods would be apparently to a person of ordinary skill in the art, and examples of such methods are described in, e.g., Machutta et al., Nat. Commun. (2017) 8:16081; Chan et al., Curr. Opin. Chem. Biol. (2015) 26:55-61; Lim, C&EN, (2017) 95 (29):10; Amigo et al., SLAS Discov. (2018) 23(5):397-404; Tian et al., MedChemComm. (2016) 7(7): 1316-1322; See Satz, CS Comb. Sci. (2016) 18 (7):415-424 each of which is hereby incorporated by reference in its entirety.

Tables 5-7 below show all the Rapafucin molecules in the present disclosure, the structural moieties are shown according to Formula (XV) An example of the chemical structure generated from Formula (XV) for compound 1 is shown below. In the case of amino acid monomers and FKBDs, a dehydration reaction occurs resulting in a peptide bond. Examples that do not designate a monomer 4 are Rapafucins composed of an FKBD with linker and only 3 monomers.

FKBD is:

-   -   Linker is:

TABLE 5 Rapafucin compound 1 to compound 578 in this disclosure. FKBD Compound with Monomer Monomer Monomer Monomer Retention Rel. Prolif., No. linkers 1 2 3 4 Time A549  1 eFKBD ra147 ra567 ra562 g 4.33 low  2 eFKBD ra147 ra566 ra562 g 4.35 low  3 eFKBD ra147 ra58 ra562 g 4.37 low  4 eFKBD ra147 ra512 ra562 g 4.32 low  5 eFKBD ra147 ra71 ra562 g 4.19 low  6 eFKBD ra147 ra135 ra562 g 4.40 low  7 eFKBD ra147 ra97 ra562 g 4.41 low  8 eFKBD ra147 y ra562 g 3.81 low  9 eFKBD ma napA ra562 g 4.71 low  10 eFKBD ra147 ra94 ra562 g 4.39 low  11 eFKBD ra147 ra137 ra562 g 4.38 low  12 eFKBD ra147 ra98 ra562 g 4.48 low  13 eFKBD ra147 ra73 ra562 g 4.40 low  14 eFKBD ra147 ra60 ra562 g 4.43 low  15 eFKBD ra147 ra353 ra562 g 4.53 low  16 eFKBD ra147 ra133 ra562 g 3.91 low  17 eFKBD ra147 ra96 ra562 g 4.47 low  18 eFKBD ra147 ra95 ra562 g 4.45 low  19 eFKBD ra147 ra70 ra562 g 4.48 low  20 eFKBD ra147 ra91 ra562 g 3.51 low  21 eFKBD ra147 ra90 ra562 g 3.44 low  22 eFKBD ra147 ra89 ra562 g 3.38 low  23 eFKBD ra147 ra301 ra562 g 3.89 low  24 eFKBD ra147 ra68 ra562 g 4.12 low  25 eFKBD ra147 ra67 ra562 g 4.13 low  26 eFKBD ra147 ra189 ra562 g 4.11 low  27 eFKBD ra147 ra144 ra562 g 4.19 low  28 eFKBD ra147 ra530 ra562 g 4.31 low  29 eFKBD ra147 cha ra562 g 4.48 low  30 eFKBD ra147 ra527 ra562 g 4.55 low  31 eFKBD ra147 ra549 ra562 g 4.59 low  32 eFKBD ra147 ra59 ra562 g 4.66 low  33 eFKBD ra147 tle ra562 g 4.23 low  34 eFKBD ra147 ra83 ra562 g 4.31 low  35 eFKBD ra147 ra533 ra562 g 4.39 low  36 eFKBD ra147 ra84 ra562 g 4.40 low  37 eFKBD ra147 ra129 ra562 g 4.69 low  38 eFKBD ra147 ra602 ra562 g 4.28 low  39 eFKBD ra147 ra122 ra562 g 4.41 low  40 eFKBD ra147 ra128 ra562 g 4.29 low  41 eFKBD ra147 ra600 ra562 g 4.29 low  42 eFKBD ra147 df ra562 g 4.30 low  43 eFKBD ra147 ra134 ra562 g 4.39 low  44 eFKBD ra147 mf ra562 g 4.45 low  45 eFKBD ra147 ra185 ra562 g 4.31 low  46 eFKBD ra147 ra124 ra562 g 4.25 low  47 eFKBD ra147 ra113 ra562 g 4.22 low  48 eFKBD ra147 ra114 ra562 g 4.17 low  49 eFKBD ra147 ra112 ra562 g 4.14 low  50 eFKBD ra147 ra87 ra562 g 4.38 low  51 eFKBD ra147 ra104 ra562 g 4.42 low  52 eFKBD ra147 ra63 ra562 g 4.43 low  53 eFKBD ma ra107 ra562 g 4.51 medium  54 eFKBD ma ra110 ra209 g 4.22 high  55 eFKBD ra147 ra119 ra562 g 4.26 low  56 eFKBD ra147 ra118 ra562 g 4.24 low  57 eFKBD ma ra110 ra562 g 4.32 high  58 eFKBD ra147 ra65 ra562 g 4.34 low  59 eFKBD ra147 ra115 ra562 g 4.34 low  60 eFKBD ra147 ra117 ra562 g 4.40 low  61 eFKBD ra147 ra116 ra562 g 4.35 low  62 eFKBD ra147 ra62 ra562 g 4.49 low  63 eFKBD ra147 ra56 ra562 g 4.54 low  64 eFKBD ra147 ra55 ra562 g 4.52 low  65 eFKBD ra147 ra366 ra562 g 4.47 low  66 eFKBD ma ra111 ra562 g 3.57 low  67 eFKBD ra147 ra109 ra562 g 3.75 low  68 eFKBD ra147 ra525 ra562 g 4.34 low  69 eFKBD ra147 ra526 ra562 g 4.37 low  70 eFKBD ra147 ra523 ra562 g 4.93 low  71 eFKBD ra147 ra521 ra562 g 4.90 low  72 eFKBD ra147 oic ra562 g 4.34 low  73 eFKBD ra147 ra102 ra562 g 4.33 low  74 eFKBD ra147 tic ra562 g 4.26 low  75 eFKBD ma ra121 ra562 g 3.96 high  76 eFKBD ra147 ra105 ra562 g 4.00 low  77 eFKBD ma ra123 ra562 g 4.47 low  78 eFKBD ma ra567 ra562 g 4.58 low  79 eFKBD ma ra566 ra562 g 4.63 low  80 eFKBD ma ra167 ra562 g 4.43 low  81 eFKBD ma ra71 ra562 g 4.40 low  82 eFKBD ma ra78 ra562 g 4.42 low  83 eFKBD ma ra327 ra562 g 3.66 low  84 eFKBD ma ra324 ra562 g 3.62 low  85 eFKBD ma rbphe ra562 g 4.22 low  86 eFKBD ma ra135 ra562 g 4.69 low  87 eFKBD ma ra97 ra562 g 4.66 low  88 eFKBD ma y ra562 g 3.89 low  89 eFKBD ma ra127 ra562 g 4.21 low  90 eFKBD ma ra171 ra562 g 4.33 low  91 eFKBD ma ra175 ra562 g 5.39 low  92 eFKBD ma ra137 ra562 g 4.65 low  93 eFKBD ma ra94 ra562 g 4.65 low  94 eFKBD ma ra98 ra562 g 4.86 low  95 eFKBD ma ra73 ra562 g 4.70 low  96 eFKBD ma ra60 ra562 g 4.71 low  97 eFKBD ma ra353 ra562 g 4.90 low  98 eFKBD ma ra133 ra562 g 3.92 low  99 eFKBD ma ra96 ra562 g 4.74 low 100 eFKBD ma ra95 ra562 g 4.73 low 101 eFKBD ma ra70 ra562 g 4.74 low 102 eFKBD ma ra491 ra562 g 3.47 low 103 eFKBD ma ra91 ra562 g 3.51 low 104 eFKBD ma ra90 ra562 g 3.41 low 105 eFKBD ma ra89 ra562 g 3.34 low 106 eFKBD ma ra301 ra562 g 3.90 low 107 eFKBD ma ra68 ra562 g 4.19 low 108 eFKBD ma ra67 ra562 g 4.19 low 109 eFKBD ma ra347 ra562 g 4.35 low 110 eFKBD ma ra189 ra562 g 4.19 low 111 eFKBD ma ra144 ra562 g 4.21 low 112 eFKBD ma ra530 ra562 g 4.40 low 113 eFKBD ma ra509 ra562 g 4.52 low 114 eFKBD ma ra507 ra562 g 4.56 low 115 eFKBD ma cha ra562 g 4.67 low 116 eFKBD ma ra527 ra562 g 4.72 low 117 eFKBD ma ra549 ra562 g 4.88 low 118 eFKBD ma ra59 ra562 g 4.94 low 119 eFKBD ma tle ra562 g 4.34 low 120 eFKBD ma ra83 ra562 g 4.40 low 121 eFKBD ma ra75 ra562 g 4.53 low 122 eFKBD ma ra533 ra562 g 4.54 low 123 eFKBD ma ra84 ra562 g 4.51 low 124 eFKBD ma ra129 ra562 g 4.89 low 125 eFKBD ma ra602 ra562 g 4.24 low 126 eFKBD ma ra122 ra562 g 4.41 low 127 eFKBD ma ra450 ra562 g 3.95 low 128 eFKBD ma ra522 ra562 g 3.83 low 129 eFKBD ma ra128 ra562 g 4.20 low 130 eFKBD ma ra600 ra562 g 4.21 low 131 eFKBD ma ra76 ra562 g 4.20 low 132 eFKBD ma df ra562 g 4.34 low 133 eFKBD ma ra134 ra562 g 4.41 low 134 eFKBD ma mf ra562 g 4.58 low 135 eFKBD ma ra185 ra562 g 4.37 low 136 eFKBD ma ra124 ra562 g 4.34 low 137 eFKBD ma ra513 ra562 g 3.99 low 138 eFKBD ma ra113 ra562 g 4.27 low 139 eFKBD ma ra114 ra562 g 4.24 low 140 eFKBD ma ra112 ra562 g 4.20 low 141 eFKBD ma ra87 ra562 g 4.49 low 142 eFKBD ma ra104 ra562 g 4.50 low 143 eFKBD ma ra148 ra562 g 4.13 low 144 eFKBD ma ra63 ra562 g 4.64 low 145 eFKBD ma ra561 ra562 g 4.62 low 146 eFKBD ma ra208 ra562 g 4.64 low 147 eFKBD ma ra382 ra562 g 4.39 low 148 eFKBD ma ra495 ra562 g 4.64 low 149 eFKBD ma ra64 ra562 g 4.46 low 150 eFKBD ma ra119 ra562 g 4.39 low 151 eFKBD ma ra118 ra562 g 4.37 low 152 eFKBD ma ra65 ra562 g 4.44 low 153 eFKBD ma ra66 ra562 g 4.73 low 154 eFKBD ma ra115 ra562 g 4.49 low 155 eFKBD ma ra117 ra562 g 4.55 low 156 eFKBD ma ra116 ra562 g 4.54 low 157 eFKBD ma ra62 ra562 g 4.76 low 158 eFKBD ma ra56 ra562 g 4.76 low 159 eFKBD ma ra534 ra562 g 4.72 medium 160 eFKBD ma ra88 ra562 g 4.28 low 161 eFKBD ma ra55 ra562 g 4.73 low 162 eFKBD ma ra366 ra562 g 4.77 low 163 eFKBD ra199 napA ra562 g 4.11 low 164 eFKBD ma ra92 ra562 g 4.56 low 165 eFKBD ra202 napA ra562 g 4.17 low 166 eFKBD ra484 napA ra562 g 4.21 low 167 eFKBD ma ra93 ra144 g 3.90 medium 168 eFKBD ml ra167 ra562 g 4.32 low 169 eFKBD ra207 ra167 ra562 g 4.28 low 170 eFKBD ra565 ra167 ra562 g 4.21 low 171 eFKBD ra172 ra167 ra562 g 4.24 low 172 eFKBD ra562 ra167 ra562 g 4.33 low 173 eFKBD ra209 ra167 ra562 g 4.28 low 174 eFKBD ra61 ra167 ra562 g 4.17 low 175 eFKBD ra74 ra167 ra562 g 4.08 low 176 eFKBD ra147 ra332 ra562 g 4.54 low 177 eFKBD ma ra332 ra562 g 4.24 low 178 eFKBD ra199 ra332 ra562 g 4.22 low 179 eFKBD ra201 ra332 ra562 g 4.30 low 180 eFKBD ra202 ra332 ra562 g 4.30 low 181 eFKBD ra203 ra332 ra562 g 4.32 low 182 eFKBD ra484 ra332 ra562 g 4.30 low 183 eFKBD ra379 ra332 ra562 g 4.41 low 184 eFKBD ml ra109 ra562 g 3.69 low 185 eFKBD ra207 ra109 ra562 g 3.67 low 186 eFKBD ra565 ra109 ra562 g 3.60 low 187 eFKBD ra562 ra109 ra562 g 3.72 low 188 eFKBD ra209 ra109 ra562 g 3.71 low 189 eFKBD ra61 ra109 ra562 g 3.59 low 190 eFKBD ra74 ra109 ra562 g 3.48 low 191 eFKBD ma ra108 ra562 g 3.21 low 192 eFKBD ra199 ra108 ra562 g 3.23 low 193 eFKBD ra201 ra108 ra562 g 3.31 low 194 eFKBD ra202 ra108 ra562 g 3.33 low 195 eFKBD ra203 ra108 ra562 g 3.36 low 196 eFKBD ra484 ra108 ra562 g 3.36 low 197 eFKBD ra379 ra108 ra562 g 3.47 low 198 eFKBD ml oic ra562 g 4.25 low 199 eFKBD ra207 oic ra562 g 4.29 low 200 eFKBD ra565 oic ra562 g 4.21 low 201 eFKBD ra172 oic ra562 g 4.23 low 202 eFKBD ra562 oic ra562 g 4.23 low 203 eFKBD ra209 oic ra562 g 4.27 low 204 eFKBD ra61 oic ra562 g 4.14 low 205 eFKBD ra74 oic ra562 g 4.07 low 206 eFKBD ra147 ra542 ra562 g 4.25 low 207 eFKBD ma ra542 ra562 g 3.92 low 208 eFKBD ra199 ra542 ra562 g 3.91 low 209 eFKBD ra201 ra542 ra562 g 4.00 low 210 eFKBD ra202 ra542 ra562 g 3.99 low 211 eFKBD ra203 ra542 ra562 g 3.98 low 212 eFKBD ra484 ra542 ra562 g 4.01 low 213 eFKBD ra379 ra542 ra562 g 4.13 low 214 eFKBD ml tic ra562 g 4.19 low 215 eFKBD ra207 tic ra562 g 4.26 low 216 eFKBD ra565 tic ra562 g 4.14 low 217 eFKBD ra172 tic ra562 g 4.16 low 218 eFKBD ra562 tic ra562 g 4.17 low 219 eFKBD ra209 tic ra562 g 4.20 low 220 eFKBD ra61 tic ra562 g 4.06 low 221 eFKBD ra74 tic ra562 g 4.02 low 222 eFKBD ma ra93 ra209 g 4.06 medium 223 eFKBD ma ra136 ra562 g 3.54 low 224 eFKBD ra199 ra136 ra562 g 3.57 low 225 eFKBD ra201 ra136 ra562 g 3.62 low 226 eFKBD ra202 ra136 ra562 g 3.64 low 227 eFKBD ra203 ra136 ra562 g 3.66 low 228 eFKBD ra484 ra136 ra562 g 3.64 low 229 eFKBD ra379 ra136 ra562 g 3.78 low 230 eFKBD ml ra545 ra562 g 4.19 low 231 eFKBD ra207 ra545 ra562 g 4.12 low 232 eFKBD ra565 ra545 ra562 g 4.10 low 233 eFKBD ra172 ra545 ra562 g 4.11 low 234 eFKBD ra562 ra545 ra562 g 4.15 low 235 eFKBD ra209 ra545 ra562 g 4.18 low 236 eFKBD ra61 ra545 ra562 g 4.08 medium 237 eFKBD ra74 ra545 ra562 g 4.02 low 238 eFKBD ra147 ra350 ra562 g 4.18 low 239 eFKBD ma ra350 ra562 g 3.87 low 240 eFKBD ra199 ra350 ra562 g 3.93 low 241 eFKBD ra201 ra350 ra562 g 3.96 low 242 eFKBD ra202 ra350 ra562 g 3.97 low 243 eFKBD ra203 ra350 ra562 g 3.97 low 244 eFKBD ra484 ra350 ra562 g 4.05 low 245 eFKBD ra379 ra350 ra562 g 4.17 low 246 eFKBD ml ra351 ra562 g 4.31 low 247 eFKBD ra207 ra351 ra562 g 4.14 low 248 eFKBD ra565 ra351 ra562 g 4.16 low 249 eFKBD ra172 ra351 ra562 g 4.19 low 250 eFKBD ra562 ra351 ra562 g 4.25 low 251 eFKBD ra209 ra351 ra562 g 4.27 low 252 eFKBD ra61 ra351 ra562 g 4.18 low 253 eFKBD ra74 ra351 ra562 g 4.02 low 254 eFKBD ma ra93 ra562 g 4.58 low 255 eFKBD ml ra93 ra562 g 4.96 low 256 eFKBD ra344 ra102 ra562 g 4.48 low 257 eFKBD ra209 ra102 ra562 g 3.24 low 258 eFKBD ra147 ra554 ra562 g 4.96 low 259 eFKBD ma ra554 ra562 g 4.49 low 260 eFKBD ra201 ra554 ra562 g 4.57 low 261 eFKBD ra203 ra554 ra562 g 4.65 low 262 eFKBD ra344 ra546 ra562 g 4.60 low 263 eFKBD ml ra546 ra562 g 4.86 low 264 eFKBD ra565 ra546 ra562 g 4.63 low 265 eFKBD ra209 ra546 ra562 g 4.78 low 266 eFKBD ra147 mw ra562 g 4.68 low 267 eFKBD ma mw ra562 g 4.37 low 268 eFKBD ra201 mw ra562 g 4.44 low 269 eFKBD ra203 mw ra562 g 4.44 low 270 eFKBD ra344 ra354 ra562 g 4.68 low 271 eFKBD ml ra354 ra562 g 4.83 low 272 eFKBD ra565 ra354 ra562 g 4.67 low 273 eFKBD ra209 ra354 ra562 g 4.80 low 274 eFKBD ra147 ra385 ra562 g 4.89 low 275 eFKBD ma ra385 ra562 g 4.45 low 276 eFKBD ra201 ra385 ra562 g 4.54 low 277 eFKBD ra203 ra385 ra562 g 4.57 low 278 eFKBD ra344 ra486 ra562 g 5.86 low 279 eFKBD ml ra486 ra562 g 5.40 low 280 eFKBD ra565 ra486 ra562 g 5.26 low 281 eFKBD ra209 ra486 ra562 g 4.29 low 282 eFKBD ra147 ra487 ra562 g 4.34 low 283 eFKBD ma ra487 ra562 g 3.94 low 284 eFKBD ra201 ra487 ra562 g 4.03 low 285 eFKBD ra203 ra487 ra562 g 4.07 low 286 eFKBD ma ra323 ra562 g 3.20 low 287 eFKBD ra201 ra323 ra562 g 5.30 low 288 eFKBD ra203 ra323 ra562 g 5.27 low 289 eFKBD ra344 ra347 ra562 g 4.56 low 290 eFKBD ml ra347 ra562 g 4.71 low 291 eFKBD ra565 ra347 ra562 g 4.55 low 292 eFKBD ra209 ra347 ra562 g 4.69 low 293 eFKBD ra147 napa ra209 g 4.29 medium 294 eFKBD ra201 ra88 ra562 g 4.35 low 295 eFKBD ra203 ra88 ra562 g 4.39 low 296 eFKBD ra344 ra137 ra562 g 4.90 low 297 eFKBD ml ra137 ra562 g 5.06 low 298 eFKBD ra565 ra137 ra562 g 4.89 low 299 eFKBD ra209 ra137 ra562 g 5.03 low 300 eFKBD ra147 ra495 ra562 g 5.05 low 301 eFKBD ra201 ra495 ra562 g 4.72 low 302 eFKBD ra203 ra495 ra562 g 4.76 low 303 eFKBD ra344 ra171 ra562 g 4.53 low 304 eFKBD ml ra171 ra562 g 4.69 low 305 eFKBD ra565 ra171 ra562 g 4.53 low 306 eFKBD ra209 ra171 ra562 g 4.66 low 307 eFKBD ra201 ra123 ra562 g 4.56 low 308 eFKBD ra203 ra123 ra562 g 4.59 low 309 eFKBD ra344 ra93 ra562 g 4.81 low 310 eFKBD ra565 ra93 ra562 g 4.77 low 311 eFKBD ra209 ra93 ra562 g 4.91 low 312 eFKBD ra147 ra107 ra549 g 4.57 medium 313 eFKBD ra201 ra64 ra562 g 4.52 low 314 eFKBD ra203 ra64 ra562 g 4.57 low 315 eFKBD ra344 ra116 ra562 g 4.78 low 316 eFKBD ml ra116 ra562 g 4.91 low 317 eFKBD ra565 ra116 ra562 g 4.82 low 318 eFKBD ra209 ra116 ra562 g 4.92 low 319 eFKBD ra147 ra107 ra562 g 4.44 low 320 eFKBD ra201 ra66 ra562 g 4.82 low 321 eFKBD ra203 ra66 ra562 g 4.88 low 322 eFKBD ra344 ra75 ra562 g 4.89 low 323 eFKBD ml ra75 ra562 g 5.04 low 324 eFKBD ra565 ra75 ra562 g 4.83 low 325 eFKBD ra209 ra75 ra562 g 4.87 low 326 eFKBD ra147 ra108 ra562 g 3.68 low 327 eFKBD ra201 ra127 ra562 g 4.31 low 328 eFKBD ra203 ra127 ra562 g 4.34 low 329 eFKBD ra344 ra113 ra562 g 4.46 low 330 eFKBD ml ra113 ra562 g 4.60 low 331 eFKBD ra565 ra113 ra562 g 4.48 low 332 eFKBD ra209 ra113 ra562 g 4.61 low 333 eFKBD ra147 ra497 ra562 g 4.24 low 334 eFKBD ra147 ra148 ra562 g 4.39 medium 335 eFKBD ra147 ra110 ra562 g 4.61 medium 336 eFKBD ra147 ra111 ra562 g 3.86 low 337 eFKBD ra147 ra121 ra549 g 4.41 medium 338 eFKBD ra147 ra121 ra562 g 4.25 low 339 eFKBD ra147 napa ra206 g 4.05 medium 340 eFKBD ra147 ra497 ra206 g 3.91 medium 341 eFKBD ra147 ra93 ra206 g 4.08 medium 342 eFKBD ra147 ra204 ra206 g 3.91 medium 343 eFKBD ra147 ra148 ra206 g 4.06 medium 344 eFKBD ra147 ra121 ra206 g 3.88 medium 345 eFKBD ra147 ra107 ra206 g 4.07 medium 346 eFKBD ra147 ra110 ra206 g 4.26 medium 347 eFKBD ra147 ra88 ra206 g 3.85 medium 348 eFKBD ra147 ra92 ra206 g 4.02 medium 349 eFKBD ra147 ra111 ra206 g 3.61 medium 350 eFKBD ra147 ra123 ra562 g 4.28 low 351 eFKBD ra147 ra93 ra209 g 4.33 medium 352 eFKBD ra147 ra204 ra209 g 4.17 low 353 eFKBD ra147 ra148 ra209 g 4.31 medium 354 eFKBD ra147 ra121 ra209 g 4.17 medium 355 eFKBD ra147 ra107 ra209 g 4.33 medium 356 eFKBD ra147 ra110 ra209 g 4.49 medium 357 eFKBD ra147 ra88 ra209 g 4.11 low 358 eFKBD ra147 ra92 ra209 g 4.28 medium 359 eFKBD ra147 ra111 ra209 g 3.86 low 360 eFKBD ra147 napa ra106 g 4.16 low 361 eFKBD ra147 ra497 ra106 g 4.06 low 362 eFKBD ra147 ra93 ra106 g 4.20 low 363 eFKBD ra147 ra204 ra106 g 4.06 low 364 eFKBD ra147 ra148 ra106 g 4.17 low 365 eFKBD ra147 ra121 ra106 g 4.02 low 366 eFKBD ra147 ra107 ra106 g 4.19 low 367 eFKBD ra147 ra110 ra106 g 4.36 low 368 eFKBD ra147 ra88 ra106 g 3.97 low 369 eFKBD ra147 ra92 ra106 g 4.15 low 370 eFKBD ra147 ra111 ra106 g 3.74 low 371 eFKBD ra147 napa ra189 g 4.17 low 372 eFKBD ra147 ra497 ra189 g 4.06 low 373 eFKBD ra147 ra93 ra189 g 4.20 low 374 eFKBD ra147 ra204 ra189 g 4.06 low 375 eFKBD ra147 ra148 ra189 g 4.18 low 376 eFKBD ra147 ra121 ra189 g 4.02 low 377 eFKBD ra147 ra107 ra189 g 4.20 low 378 eFKBD ra147 ra110 ra189 g 4.36 low 379 eFKBD ra147 ra88 ra189 g 3.98 low 380 eFKBD ra147 ra92 ra189 g 4.16 low 381 eFKBD ra147 ra111 ra189 g 3.74 low 382 eFKBD ra147 napa ra144 g 4.17 low 383 eFKBD ra147 ra497 ra144 g 4.03 low 384 eFKBD ra147 ra93 ra144 g 4.19 low 385 eFKBD ra147 ra204 ra144 g 4.07 low 386 eFKBD ra147 ra121 ra144 g 4.03 medium 387 eFKBD ra147 ra107 ra144 g 4.19 low 388 eFKBD ra147 ra110 ra144 g 4.39 medium 389 eFKBD ra147 ra88 ra144 g 3.96 low 390 eFKBD ra147 ra92 ra144 g 4.16 medium 391 eFKBD ra147 ra111 ra144 g 3.73 low 392 eFKBD ra147 napa ra126 g 4.00 low 393 eFKBD ra147 ra497 ra126 g 3.84 low 394 eFKBD ra147 ra93 ra126 g 4.03 low 395 eFKBD ra147 ra511 ra126 g 4.03 low 396 eFKBD ra147 ra204 ra126 g 3.87 low 397 eFKBD ra147 ra148 ra126 g 4.00 low 398 eFKBD ra147 ra121 ra126 g 3.83 low 399 eFKBD ra147 ra107 ra126 g 4.03 low 400 eFKBD ra147 ra110 ra126 g 4.18 low 401 eFKBD ra147 ra88 ra126 g 3.77 low 402 eFKBD ra147 ra92 ra126 g 3.98 low 403 eFKBD ra147 ra111 ra126 g 3.51 low 404 eFKBD ra147 napa ra549 g 4.54 low 405 eFKBD ra147 ra127 ra562 g 4.22 low 406 eFKBD ra147 ra93 ra549 g 4.58 low 407 eFKBD ra147 ra204 ra549 g 4.43 low 408 eFKBD ra147 ra148 ra549 g 4.55 medium 409 eFKBD ra147 ra136 ra562 g 4.00 low 410 eFKBD ra147 ra110 ra549 g 4.78 medium 411 eFKBD ra147 ra88 ra549 g 4.34 medium 412 eFKBD ra147 ra92 ra549 g 4.53 medium 413 eFKBD ra147 ra111 ra549 g 4.15 low 414 eFKBD ma ra497 ra562 g 3.94 low 415 eFKBD ra147 ra148 ra144 g 4.18 medium 416 eFKBD ra147 ra497 ra209 g 4.17 medium 417 eFKBD ra147 ra497 ra549 g 4.43 medium 418 eFKBD ra147 ra64 ra562 g 4.32 low 419 eFKBD ma ra497 ra206 g 3.57 low 420 eFKBD ma ra93 ra206 g 3.80 low 421 eFKBD ma ra204 ra206 g 3.57 low 422 eFKBD ma ra148 ra206 g 3.74 low 423 eFKBD ma ra121 ra206 g 3.53 low 424 eFKBD ma ra107 ra206 g 3.79 low 425 eFKBD ma ra110 ra206 g 3.96 low 426 eFKBD ma ra88 ra206 g 3.49 low 427 eFKBD ma ra92 ra206 g 3.72 low 428 eFKBD ma napa ra209 g 4.04 low 429 eFKBD ma ra497 ra209 g 3.91 medium 430 eFKBD ma ra204 ra209 g 3.91 low 431 eFKBD ma ra148 ra209 g 4.04 low 432 eFKBD ma ra107 ra209 g 4.06 low 433 eFKBD ra147 ra66 ra562 g 4.49 low 434 eFKBD ma ra88 ra209 g 3.83 medium 435 eFKBD ma napa ra106 g 3.90 low 436 eFKBD ma ra497 ra106 g 3.75 low 437 eFKBD ma ra93 ra106 g 3.93 low 438 eFKBD ma ra204 ra106 g 3.74 low 439 eFKBD ma ra148 ra106 g 3.91 low 440 eFKBD ma ra121 ra106 g 3.72 low 441 eFKBD ma ra107 ra106 g 3.93 low 442 eFKBD ma ra110 ra106 g 4.10 low 443 eFKBD ma ra88 ra106 g 3.66 low 444 eFKBD ma ra92 ra106 g 3.90 low 445 eFKBD ma ra111 ra106 g 3.35 low 446 eFKBD ma napa ra189 g 3.86 low 447 eFKBD ma ra497 ra189 g 3.71 low 448 eFKBD ma ra93 ra189 g 3.90 low 449 eFKBD ma ra204 ra189 g 3.72 low 450 eFKBD ma ra148 ra189 g 3.86 low 451 eFKBD ma ra121 ra189 g 3.67 low 452 eFKBD ma ra107 ra189 g 3.90 low 453 eFKBD ma ra110 ra189 g 4.07 low 454 eFKBD ma ra88 ra189 g 3.65 low 455 eFKBD ma ra92 ra189 g 3.85 low 456 eFKBD ma ra111 ra189 g 3.33 low 457 eFKBD ma napa ra144 g 3.87 low 458 eFKBD ma ra497 ra144 g 3.70 medium 459 eFKBD ma ra204 ra144 g 3.69 low 460 eFKBD ma ra148 ra144 g 3.88 low 461 eFKBD ma ra121 ra144 g 3.70 medium 462 eFKBD ma ra107 ra144 g 3.91 low 463 eFKBD ma ra110 ra144 g 4.08 medium 464 eFKBD ma ra88 ra144 g 3.63 low 465 eFKBD ma ra92 ra144 g 3.87 low 466 eFKBD ma ra111 ra144 g 3.30 low 467 eFKBD ma ra497 ra126 g 3.46 low 468 eFKBD ma ra148 ra126 g 3.67 low 469 eFKBD ma ra121 ra126 g 3.44 low 470 eFKBD ma ra107 ra126 g 3.72 low 471 eFKBD ma ra110 ra126 g 3.89 low 472 eFKBD ma ra92 ra126 g 3.69 low 473 eFKBD ma ra111 ra126 g 3.04 low 474 eFKBD ma napa ra549 g 4.29 low 475 eFKBD ma ra497 ra549 g 4.16 low 476 eFKBD ma ra93 ra549 g 4.31 low 477 eFKBD ma ra204 ra549 g 4.14 low 478 eFKBD ma ra148 ra549 g 4.31 low 479 eFKBD ma ra121 ra549 g 4.15 low 480 eFKBD ma ra107 ra549 g 4.32 low 481 eFKBD ma ra110 ra549 g 4.51 low 482 eFKBD ma ra88 ra549 g 4.09 low 483 eFKBD ma ra92 ra549 g 4.29 low 484 eFKBD ma ra111 ra549 g 3.86 low 485 eFKBD ra147 ra88 ra562 g 4.13 low 486 eFKBD ml napa ra549 g 5.13 low 487 eFKBD ml napa ra144 g 4.53 low 488 eFKBD mi napa ra562 g 4.85 low 489 eFKBD mi napa ra549 g 5.10 low 490 eFKBD mv napa ra209 g 4.56 low 491 eFKBD ra379 napa ra549 g 4.96 low 492 eFKBD ra379 napa ra144 g 4.40 low 493 eFKBD ra203 ra185 ra209 g 4.31 low 494 eFKBD ra202 ra185 ra209 g 4.48 low 495 eFKBD ra310 ra185 ra209 g 4.68 low 496 eFKBD ra203 ra110 ra562 g 4.95 low 497 eFKBD ra202 ra110 ra562 g 4.91 low 498 eFKBD ra310 ra110 ra562 g 5.32 low 499 eFKBD ra203 ra93 ra209 g 4.46 low 500 eFKBD ra202 ra93 ra209 g 4.47 low 501 eFKBD ra310 ra93 ra209 g 4.80 low 502 eFKBD ra147 ra92 ra562 g 4.41 low 503 eFKBD mi ra497 ra209 g 4.54 low 504 eFKBD mi ra497 ra549 g 4.93 low 505 eFKBD mi ra497 ra144 g 4.32 low 506 eFKBD ra379 ra497 ra562 g 4.48 low 507 eFKBD ra379 ra497 ra209 g 4.40 low 508 eFKBD ra379 ra497 ra549 g 4.78 low 509 eFKBD ra379 ra497 ra144 g 4.20 low 510 eFKBD ra147 ra93 ra562 g 4.44 low 511 eFKBD ml ra93 ra549 g 5.05 low 512 eFKBD ra201 napA ra562 g 4.15 low 513 eFKBD mi ra93 ra562 g 4.83 low 514 eFKBD mi ra93 ra209 g 4.66 low 515 eFKBD mi ra93 ra549 g 5.06 low 516 eFKBD mi ra93 ra144 g 4.47 low 517 eFKBD ra379 ra93 ra562 g 4.70 low 518 eFKBD ra379 ra93 ra209 g 4.56 low 519 eFKBD ra379 ra93 ra549 g 4.91 low 520 eFKBD ra379 ra93 ra144 g 4.37 low 521 eFKBD ml ra148 ra562 g 4.91 low 522 eFKBD ml ra148 ra209 g 4.73 low 523 eFKBD ml ra148 ra549 g 5.17 low 524 eFKBD mi ra148 ra562 g 4.86 low 525 eFKBD mi ra148 ra209 g 4.69 low 526 eFKBD mi ra148 ra549 g 5.12 low 527 eFKBD mi ra148 ra144 g 4.51 low 528 eFKBD ra379 ra148 ra562 g 4.74 low 529 eFKBD ra379 ra148 ra209 g 4.59 low 530 eFKBD ra379 ra148 ra549 g 4.98 low 531 eFKBD ra379 ra148 ra144 g 4.40 low 532 eFKBD ra203 napA ra562 g 4.18 low 533 eFKBD ml ra107 ra209 g 4.72 low 534 eFKBD ml ra107 ra144 g 4.52 low 535 eFKBD mi ra107 ra562 g 4.83 low 536 eFKBD mi ra107 ra209 g 4.69 low 537 eFKBD mi ra107 ra549 g 5.09 low 538 eFKBD mi ra107 ra144 g 4.49 low 539 eFKBD ra379 ra107 ra562 g 4.73 low 540 eFKBD ra379 ra107 ra209 g 4.57 low 541 eFKBD ra379 ra107 ra549 g 4.94 low 542 eFKBD ra379 ra107 ra144 g 4.40 low 543 eFKBD ml ra121 ra562 g 4.64 low 544 eFKBD ml ra121 ra209 g 4.49 low 545 eFKBD ra379 napA ra562 g 4.30 low 546 eFKBD ml ra121 ra144 g 4.33 low 547 eFKBD mi ra121 ra562 g 4.60 low 548 eFKBD mi ra121 ra209 g 4.48 low 549 eFKBD mi ra121 ra549 g 4.86 low 550 eFKBD mi ra121 ra144 g 4.30 low 551 eFKBD ra379 ra121 ra562 g 4.48 low 552 eFKBD ra379 ra121 ra209 g 4.35 low 553 eFKBD ra379 ra121 ra549 g 4.71 low 554 eFKBD ra379 ra121 ra144 g 4.18 low 555 eFKBD ra347 ra110 ra144 g 4.98 low 556 eFKBD ra319 ra110 ra562 g 4.78 low 557 eFKBD ra319 ra110 ra209 g 4.59 low 558 eFKBD ra319 ra110 ra549 g 4.94 low 559 eFKBD ra319 ra110 ra144 g 4.44 low 560 rae1 ra147 napA ra562 g 5.56 medium 561 rae2 ra147 napA ra562 g 5.63 medium 562 rae3 ra147 napA ra562 g 5.48 medium 563 rae4 ra147 napA ra562 g 5.47 low 564 rae5 ra147 napA ra562 g 5.48 low 565 rae9 ra147 napA ra562 g 5.35 medium 566 rae10 ra147 napA ra562 g 5.10 medium 567 rae11 ra147 napA ra562 g 5.11 medium 568 rae12 ra147 napA ra562 g 5.74 medium 569 rae13 ra147 napA ra562 g 5.27 medium 570 rae14 ra147 napA ra562 g 5.72 medium 571 rae16 ra147 napA ra562 g 5.93 low 572 rae17 ra147 napA ra562 g 4.41 medium 573 rae18 ra147 napA ra562 g 5.49 low 574 rae19 ra147 napA ra562 g 5.60 low 575 eFKBD ra147 napA ra562 g 5.44 low 576 rae20 ra147 napA ra562 g 5.56 medium 577 eFKBD 2-Nal mSerBu Gly 6.45 low 578 eFKBD 2-Nal mNle Gly 6.44 low

TABLE 6 Rapafucin compound 579 to compound 877. FKBD Compound with Monomer Monomer Monomer Monomer Retention Rel. Prolif., No. linkers 1 2 3 4 Time NCI-H929 579 eFKBD mf dF sar dF 4.105 low 580 eFKBD ra208 dF sar dF 4.158 low 581 eFKBD ra561 dF sar dF 4.189 low 582 eFKBD ra531 dF sar dF 4.252 low 583 eFKBD ra382 dF sar dF 4.055 low 584 eFKBD ra537 dF sar dF 4.042 low 585 eFKBD ra577 dF sar dF 3.342 low 586 eFKBD ra450 dF sar dF 3.767 low 587 eFKBD ra522 dF sar dF 3.671 low 588 eFKBD ra513 dF sar dF 3.769 low 589 eFKBD ra509 dF sar dF 4.171 low 590 eFKBD ra507 dF sar dF 4.143 low 591 eFKBD ra534 dF sar dF 4.221 low 592 eFKBD ra578 dF sar dF 3.71 low 593 eFKBD ra523 dF sar dF 3.198 low 594 eFKBD ra521 dF sar dF 3.308 low 595 eFKBD ra520 dF sar dF 3.646 low 596 eFKBD ra549 dF sar dF 4.392 low 597 eFKBD ra600 dF sar dF 3.969 low 598 eFKBD ra551 dF sar dF 4.233 low 599 eFKBD ra518 dF sar dF 3.876 low 600 eFKBD cha dF sar dF 4.264 high 601 eFKBD ra527 dF sar dF 4.257 high 602 eFKBD ra566 dF sar dF 4.215 low 603 eFKBD ra567 dF sar dF 4.189 low 604 eFKBD ra533 dF sar dF 4.135 low 605 eFKBD ra530 dF sar dF 4.111 low 606 eFKBD ra579 dF sar dF 3.649 low 607 eFKBD ra55 dF sar dF 4.26 low 608 eFKBD ra56 dF sar dF 4.259 low 609 eFKBD tza dF sar dF 3.759 low 610 eFKBD ra58 dF sar dF 3.607 low 611 eFKBD ra59 dF sar dF 4.367 low 612 eFKBD ra60 dF sar dF 5.05 low 613 eFKBD ra61 dF sar dF 4.001 low 614 eFKBD ra62 dF sar dF 4.283 low 615 eFKBD ra63 dF sar dF 4.23 low 616 eFKBD ra64 dF sar dF 4.87 low 617 eFKBD ra65 dF sar dF 4.156 low 618 eFKBD ra66 dF sar dF 4.303 low 619 eFKBD ra67 dF sar dF 3.968 low 620 eFKBD ra68 dF sar dF 3.983 low 621 eFKBD ra69 dF sar dF 4.076 low 622 eFKBD ra70 dF sar dF 4.286 low 623 eFKBD ra71 dF sar dF 4.111 low 624 eFKBD ra73 dF sar dF 4.283 low 625 eFKBD ra74 dF sar dF 3.899 low 626 eFKBD ra75 dF sar dF 4.16 low 627 eFKBD ra76 dF sar dF 4.616 low 628 eFKBD ra511 dF sar dF 4.289 low 629 eFKBD ra78 dF sar dF 4.119 low 630 eFKBD ra79 dF sar dF 4.255 low 631 eFKBD ra83 dF sar dF 4.065 low 632 eFKBD ra84 dF sar dF 4.155 low 633 eFKBD ra87 dF sar dF 4.123 low 634 eFKBD ra88 dF sar dF 4.023 low 635 eFKBD ra89 dF sar dF 3.242 low 636 eFKBD ra90 dF sar dF 3.298 low 637 eFKBD ra91 dF sar dF 3.418 low 638 eFKBD ra92 dF sar dF 4.206 low 639 eFKBD ra93 dF sar dF 4.232 low 640 eFKBD ra94 dF sar dF 4.245 low 641 eFKBD ra95 dF sar dF 4.3 low 642 eFKBD ra96 dF sar dF 4.3 low 643 eFKBD ra97 dF sar dF 4.231 low 644 eFKBD ra98 dF sar dF 4.33 low 645 eFKBD ra353 dF sar dF 4.358 low 646 eFKBD ra104 dF sar dF 4.133 low 647 eFKBD ra106 dF sar dF 3.942 low 648 eFKBD ra107 dF sar dF 4.228 low 649 eFKBD ra108 dF sar dF 3.467 low 650 eFKBD ra110 dF sar dF 4.368 low 651 eFKBD ra111 dF sar dF 3.74 low 652 eFKBD ra112 dF sar dF 3.984 low 653 eFKBD ra113 dF sar dF 4.007 low 654 eFKBD ra114 dF sar dF 3.994 low 655 eFKBD ra115 dF sar dF 4.161 low 656 eFKBD ra116 dF sar dF 4.194 low 657 eFKBD ra117 dF sar dF 4.201 low 658 eFKBD ra119 dF sar dF 4.101 low 659 eFKBD ra120 dF sar dF 4.164 low 660 eFKBD ra121 dF sar dF 4.091 low 661 eFKBD ra123 dF sar dF 1.825 low 662 eFKBD ra124 dF sar dF 4.07 low 663 eFKBD ra126 dF sar dF 3.797 low 664 eFKBD ra127 dF sar dF 4.014 low 665 eFKBD ra128 dF sar dF 4.012 low 666 eFKBD ra132 dF sar dF 3.863 low 667 eFKBD ra135 dF sar dF 4.226 low 668 eFKBD ra144 dF sar dF 4.573 low 669 eFKBD ra148 dF sar dF 4.164 low 670 eFKBD ra171 dF sar dF 4.013 low 671 eFKBD ra173 dF sar dF 3.614 low 672 eFKBD ra175 dF sar dF 4.582 low 673 eFKBD ra176 dF sar dF 3.334 medium 674 eFKBD ra185 dF sar dF 4.055 low 675 eFKBD mf ra537 sar dF 4.129 low 676 eFKBD ra561 ra537 sar dF 4.139 low 677 eFKBD ra63 ra537 sar dF 4.182 low 678 eFKBD ra526 ra537 sar dF 4.129 low 679 eFKBD cha ra537 sar dF 4.239 low 680 eFKBD ra75 ra537 sar dF 4.145 low 681 eFKBD mf ra507 sar dF 4.218 low 682 eFKBD ra521 ra507 sar dF 3.257 low 683 eFKBD ra347 ra507 sar dF 4.142 low 684 eFKBD ra354 ra507 sar dF 4.188 low 685 eFKBD ra64 ra507 sar dF 4.202 low 686 eFKBD ra89 ra507 sar dF 0.393 low 687 eFKBD mf ra521 sar dF 3.353 medium 688 eFKBD ra561 ra521 sar dF 3.51 low 689 eFKBD ra382 ra521 sar dF 3.329 low 690 eFKBD ra513 ra521 sar dF 3.096 low 691 eFKBD ra75 ra521 sar dF 3.423 low 692 eFKBD tza ra521 sar dF 2.97 low 693 eFKBD mf ra527 sar dF 4.32 low 694 eFKBD napa ra527 sar dF 4.386 low 695 eFKBD cha ra527 sar dF 4.496 low 696 eFKBD ra107 ra527 sar dF 4.399 low 697 eFKBD ra63 ra527 sar dF 4.425 low 698 eFKBD ra171 ra527 sar dF 4.191 low 699 eFKBD mf ra566 sar dF 4.256 low 700 eFKBD ra521 ra566 sar dF 3.42 low 701 eFKBD ra347 ra566 sar dF 4.179 low 702 eFKBD ra107 ra566 sar dF 4.331 low 703 eFKBD ra64 ra566 sar dF 4.102 low 704 eFKBD tza ra566 sar dF 3.929 low 705 eFKBD mf napa sar dF 4.254 low 706 eFKBD napa napa sar dF 4.311 low 707 eFKBD cha napa sar dF 4.383 low 708 eFKBD ra354 napa sar dF 4.232 low 709 eFKBD ra171 napa sar dF 4.167 low 710 eFKBD ra89 napa sar dF 3.46 low 711 eFKBD mf ra55 sar dF 4.326 low 712 eFKBD ra561 ra55 sar dF 4.363 low 713 eFKBD ra526 ra55 sar dF 4.283 low 714 eFKBD ra63 ra55 sar dF 4.37 low 715 eFKBD ra171 ra55 sar dF 4.159 low 716 eFKBD ra89 ra55 sar dF 3.451 low 717 eFKBD mf ra56 sar dF 4.261 low 718 eFKBD ra561 ra56 sar dF 4.343 low 719 eFKBD ra513 ra56 sar dF 3.919 low 720 eFKBD ra347 ra56 sar dF 4.202 low 721 eFKBD ra75 ra56 sar dF 4.305 low 722 eFKBD ra173 ra56 sar dF 3.822 low 723 eFKBD mf ra59 sar dF 4.381 low 724 eFKBD ra526 ra59 sar dF 4.353 low 725 eFKBD cha ra59 sar dF 4.598 low 726 eFKBD ra107 ra59 sar dF 4.514 low 727 eFKBD ra75 ra59 sar dF 4.487 low 728 eFKBD tza ra59 sar dF 4.06 low 729 eFKBD mf ra60 sar dF 4.373 low 730 eFKBD napa ra60 sar dF 4.444 low 731 eFKBD ra382 ra60 sar dF 4.338 low 732 eFKBD ra107 ra60 sar dF 4.46 low 733 eFKBD ra64 ra60 sar dF 4.358 low 734 eFKBD ra89 ra60 sar dF 3.661 low 735 eFKBD mf ra65 sar dF 4.229 low 736 eFKBD ra561 ra65 sar dF 4.288 low 737 eFKBD ra347 ra65 sar dF 4.142 low 738 eFKBD ra354 ra65 sar dF 4.185 low 739 eFKBD ra171 ra65 sar dF 4.12 low 740 eFKBD ra173 ra65 sar dF 3.776 low 741 eFKBD mf ra67 sar dF 4.046 low 742 eFKBD napa ra67 sar dF 4.144 low 743 eFKBD ra513 ra67 sar dF 3.696 low 744 eFKBD ra382 ra67 sar dF 4.009 low 745 eFKBD ra171 ra67 sar dF 3.991 low 746 eFKBD ra173 ra67 sar dF 3.56 low 747 eFKBD mf ra70 sar dF 4.417 low 748 eFKBD ra513 ra70 sar dF 4.104 low 749 eFKBD ra63 ra70 sar dF 4.504 low 750 eFKBD ra107 ra70 sar dF 4.477 low 751 eFKBD ra75 ra70 sar dF 4.461 low 752 eFKBD ra354 ra70 sar dF 4.461 low 753 eFKBD mf ra144 sar dF 4.082 low 754 eFKBD napa ra144 sar dF 4.215 low 755 eFKBD ra173 ra144 sar dF 3.611 low 756 eFKBD cha ra144 sar dF 4.216 low 757 eFKBD ra354 ra144 sar dF 4.111 low 758 eFKBD mf ra354 sar dF 4.315 low 759 eFKBD ra513 ra354 sar dF 3.942 low 760 eFKBD ra382 ra354 sar dF 4.351 low 761 eFKBD ra64 ra354 sar dF 4.354 low 762 eFKBD ra63 ra354 sar dF 4.485 low 763 eFKBD ra89 ra354 sar dF 3.554 low 764 eFKBD mf ra533 sar dF 4.273 low 765 eFKBD ra347 ra533 sar dF 4.204 low 766 eFKBD ra382 ra533 sar dF 4.252 low 767 eFKBD ra173 ra533 sar dF 3.845 low 768 eFKBD ra64 ra533 sar dF 4.325 low 769 eFKBD mf ra567 sar ra60 5.28 low 770 eFKBD mf ra537 sar ra525 4.74 low 771 eFKBD mf ra527 sar ra537 4.993 low 772 eFKBD mf ra537 sar ra566 4.871 low 773 eFKBD mf ra567 sar ra537 4.881 low 774 eFKBD mf ra537 sar ra533 4.765 low 775 eFKBD mf ra59 sar ra537 5.226 low 776 eFKBD mf ra537 sar ra60 4.989 low 777 eFKBD mf ra537 sar ra67 4.5 low 778 eFKBD mf ra70 sar ra537 5.023 low 779 eFKBD mf ra537 sar ra144 4.505 low 780 eFKBD mf ra354 sar ra537 4.749 low 781 eFKBD mf ra507 sar ra525 4.948 low 782 eFKBD mf ra507 sar ra566 5.088 low 783 eFKBD mf ra567 sar ra507 5.034 low 784 eFKBD mf ra507 sar ra533 4.97 low 785 eFKBD mf ra55 sar ra507 5.175 low 786 eFKBD mf ra507 sar ra56 5.191 low 787 eflcbd mf ra59 sar ra507 5.424 low 788 eFKBD mf ra507 sar ra60 5.184 low 789 eFKBD mf ra65 sar ra507 4.886 low 790 eFKBD mf ra67 sar ra507 4.656 low 791 eFKBD mf ra70 sar ra507 5.206 low 792 eFKBD mf ra507 sar ra144 4.666 low 793 eFKBD mf ra354 sar ra507 4.898 low 794 eFKBD mf ra566 sar ra521 3.993 low 795 eFKBD mf ra533 sar ra525 4.247 low 796 eFKBD mf ra56 sar ra521 4.04 low 797 eFKBD mf ra60 sar ra537 5.01 low 798 eFKBD mf ra67 sar ra537 4.523 low 799 eFKBD mf ra537 sar ra70 4.998 low 800 eFKBD mf ra144 sar ra537 4.516 low 801 eFKBD mf ra537 sar ra354 4.732 low 802 eFKBD mf ra566 sar ra527 5.259 low 803 eFKBD mf ra527 sar ra567 5.237 low 804 eFKBD mf ra527 sar ra55 5.356 low 805 eFKBD mf ra56 sar ra527 5.375 low 806 eFKBD mf ra527 sar ra59 5.647 low 807 eFKBD mf ra60 sar ra527 5.345 low 808 eFKBD mf ra527 sar ra65 5.033 low 809 eFKBD mf ra67 sar ra527 4.798 low 810 eFKBD mf ra70 sar ra533 5.155 low 811 eFKBD mf ra527 sar ra354 5.076 low 812 eFKBD mf ra567 sar ra566 5.11 low 813 eFKBD mf ra59 sar ra566 5.479 low 814 eFKBD mf ra566 sar ra60 5.242 low 815 eFKBD mf ra65 sar ra566 4.932 low 816 eFKBD mf ra566 sar ra67 4.716 low 817 eFKBD mf ra70 sar ra566 5.298 low 818 eFKBD mf ra566 sar ra144 4.729 low 819 eFKBD mf ra354 sar ra566 4.968 low 820 eFKBD mf ra566 sar ra533 5.027 low 821 eFKBD mf ra59 sar ra567 5.461 low 822 eFKBD mf ra65 sar ra567 4.938 low 823 eFKBD mf ra567 sar ra67 4.706 low 824 eFKBD mf ra70 sar ra567 5.267 low 825 eFKBD mf ra55 sar ra533 5.146 low 826 eFKBD mf ra59 sar ra533 5.378 low 827 eFKBD mf ra533 sar ra60 5.166 low 828 eFKBD mf ra65 sar ra533 4.851 low 829 eFKBD mf ra533 sar ra67 4.65 low 830 eFKBD mf ra533 sar ra144 4.659 low 831 eFKBD mf ra354 sar ra533 4.889 low 832 eFKBD mf ra59 sar ra55 5.603 low 833 eFKBD mf ra55 sar ra60 5.352 low 834 eFKBD mf ra65 sar ra55 5.028 low 835 eFKBD mf ra67 sar ra55 4.798 low 836 eFKBD mf ra70 sar ra55 5.382 low 837 eFKBD mf ra55 sar ra144 4.811 low 838 eFKBD mf ra59 sar ra56 5.631 low 839 eFKBD mf ra56 sar ra60 5.367 low 840 eFKBD mf ra65 sar ra56 5.049 low 841 eFKBD mf ra56 sar ra67 4.82 low 842 eFKBD mf ra70 sar ra56 5.411 low 843 eFKBD mf ra354 sar ra56 5.079 low 844 eFKBD mf ra59 sar ra60 5.553 low 845 eFKBD mf ra65 sar ra59 5.23 low 846 eFKBD mf ra70 sar ra59 5.602 low 847 eFKBD mf ra59 sar ra144 4.976 low 848 eFKBD mf ra354 sar ra59 5.25 low 849 eFKBD mf ra60 sar ra65 5.031 low 850 eFKBD mf ra67 sar ra60 4.813 low 851 eFKBD mf ra60 sar ra70 5.349 low 852 eFKBD mf ra67 sar ra65 4.54 low 853 eFKBD mf ra65 sar ra70 5.053 low 854 eFKBD mf ra144 sar ra65 4.54 low 855 eFKBD mf ra65 sar ra354 4.771 low 856 eFKBD mf ra144 sar ra55 4.77 low 857 eFKBD mf ra354 sar ra55 5.049 low 858 eFKBD mf ra70 sar ra144 4.834 low 859 eFKBD mf ra354 sar ra70 5.081 low 860 eFKBD mf ra144 sar ra354 4.574 low 861 eFKBD mf ra527 sar ra507 5.191 low 862 efkbd ra606 df sar df 5.285 high 863 rae21 ra98 df sar df 4.281 low 864 rae19 ra98 df sar df 4.22 low 865 aFKBD ra98 df sar df 4.098 low 866 eflcbd ra607 df sar df 5.077 high 867 rae21 ra492 df sar df 5.75 low 868 rae19 ra492 df sar df 5.54 low 869 aFKBD ra492 df sar df 5.403 low 870 efkbd ra608 df sar df 4.948 low 871 rae34 mf df sar df 3.854 low 872 rae35 mf df sar df 4.434 low 873 raa19 mf df sar df 4.871 low 874 raa20 mf df sar df 4.622 low 875 rae36 mf df sar df 5.43 low 876 rae27 mf df sar df 4.962 low 877 rae37 ra398 df sar df 4.181 kmv

TABLE 7 Rapafucin compound 878 to compound 1604. FKBD Rel. Compound with Monomer Monomer Monomer Monomer Retention Uptake, No. linkers 1 2 3 4 Time 293T  878 aFKBD ra104 mf dp ml 5.14 low  879 aFKBD ml p ra195 f 4.22 low  880 aFKBD ml p mf f 4.24 low  881 aFKBD ml dp ra195 f 4.33 low  882 aFKBD ra207 p ra195 f 4.33 low  883 aFKBD ml dp mf f 4.33 low  884 aFKBD ra207 p mf f 4.16 low  885 aFKBD ra207 dp ra195 f 4.10 low  886 aFKBD f ra195 p ml 4.14 low  887 aFKBD f ra195 p ra207 4.18 low  888 aFKBD f ra195 dp ml 4.13 low  889 aFKBD f mf P ml 4.05 low  890 aFKBD dF ra195 p ml 4.06 low  891 aFKBD f mf dp ml 4.14 low  892 aFKBD dF ra195 dp ml 4.11 low  893 aFKBD dF mf p ml 4.11 low  894 aFKBD ra381 mf dp ml 4.15 low  895 aFKBD ra400 mf dp ml 4.13 medium  896 aFKBD ra329 mf dp ml 4.10 medium  897 aFKBD ra325 mf dp ml 4.17 medium  898 aFKBD ra516 mf dp ml 4.27 high  899 aFKBD ra381 f dp ml 4.06 low  900 aFKBD ra400 f dp ml 4.06 low  901 aFKBD ra329 f dp ml 4.03 low  902 aFKBD ra325 f dp ml 4.11 low  903 aFKBD ra516 f dp ml 4.17 high  904 aFKBD ra522 f dp ml 3.78 low  905 aFKBD ra450 f dp ml 3.89 high  906 aFKBD ra602 f dp ml 4.04 high  907 aFKBD ra381 dF dp ml 4.07 medium  908 aFKBD ra400 dF dp ml 4.08 low  909 aFKBD ra329 dF dp ml 4.05 medium  910 aFKBD ra325 dF dp ml 4.18 medium  911 aFKBD ra516 dF dp ml 4.29 low  912 aFKBD ra522 dF dp ml 3.87 low  913 aFKBD ra450 dF dp ml 3.93 low  914 aFKBD ra602 dF dp ml 4.11 low  915 aFKBD ra381 ra195 dp ml 4.10 low  916 aFKBD ra400 ra195 dp ml 4.12 low  917 aFKBD ra329 ra195 dp ml 4.08 low  918 aFKBD ra325 ra195 dp ml 4.18 low  919 aFKBD ra516 ra195 dp ml 4.26 low  920 aFKBD ra522 ra195 dp ml 3.82 low  921 aFKBD ra450 ra195 dp ml 3.91 low  922 aFKBD ra602 ra195 dp ml 4.11 low  923 aFKBD ra381 y dp ml 3.79 low  924 aFKBD ra400 y dp ml 3.78 low  925 aFKBD ra329 y dp ml 3.76 low  926 aFKBD ra325 y dp ml 3.82 low  927 aFKBD ra516 y dp ml 3.89 high  928 aFKBD ra602 ra577 dp ml 3.45 low  929 aFKBD ra602 ra173 dp ml 3.60 low  930 aFKBD ra602 ra66 dp ml 4.29 medium  931 aFKBD ra602 ra56 dp ml 4.30 low  932 aFKBD ra602 ra64 dp ml 4.13 high  933 aFKBD ra602 ra171 dp ml 4.08 high  934 aFKBD ra602 ra63 dp ml 4.27 low  935 aFKBD ra577 mf dp ml 3.55 low  936 aFKBD ra173 mf dp ml 3.77 low  937 aFKBD ra66 mf dp ml 4.44 low  938 aFKBD ra56 mf dp ml 4.43 low  939 aFKBD ra64 mf dp ml 4.27 low  940 aFKBD ra171 mf dp ml 4.20 low  941 aFKBD ra63 mf dp ml 4.38 low  942 aFKBD ra577 y dp ml 3.23 low  943 aFKBD ra173 y dp ml 3.41 low  944 aFKBD ra66 y dp ml 4.06 high  945 aFKBD ra56 y dp ml 4.06 high  946 aFKBD ra64 y dp ml 3.93 low  947 aFKBD ra171 y dp ml 3.86 low  948 aFKBD ra63 y dp ml 4.01 low  949 aFKBD ra122 mf dp ml 4.13 low  950 aFKBD f ra512 dp ml 4.32 low  951 aFKBD y ra512 dp ml 4.08 low  952 aFKBD mf ra512 dp ml 4.44 low  953 aFKBD ra522 ra512 dp ml 4.04 low  954 aFKBD ra450 ra512 dp ml 4.12 medium  955 aFKBD ra602 ra348 dp ml 4.09 high  956 aFKBD ra602 ra547 dp ml 3.96 high  957 aFKBD ra602 ra381 dp ml 4.01 medium  958 aFKBD ra602 ra400 dp ml 4.04 low  959 aFKBD ra602 ra329 dp ml 4.03 medium  960 aFKBD ra602 ra325 dp ml 4.09 low  961 aFKBD ra602 ra516 dp ml 4.19 low  962 aFKBD ra602 mf dp ra348 4.15 low  963 aFKBD ra602 mf dp ra547 3.99 low  964 aFKBD ra602 mf dp sar 3.70 low  965 aFKBD ra602 mf dp ra147 4.16 high  966 aFKBD ra602 y dp ra348 3.73 low  967 aFKBD ra602 y dp ra547 3.60 low  968 aFKBD ra602 y dp sar 3.17 low  969 aFKBD ra602 y dp ra147 3.78 low  970 aFKBD ra602 y dp mi 3.74 medium  971 aFKBD ra512 mf dp ml 4.36 low  972 aFKBD ra602 mf dp cha 4.32 low  973 aFKBD ra602 mf dp ra84 4.24 low  974 aFKBD ra602 mf dp ra206 3.88 low  975 aFKBD ra602 mf dp ra209 4.21 low  976 aFKBD ra602 mf dp ra80 4.21 low  977 aFKBD ra602 mf dp ra549 4.57 low  978 aFKBD ra602 mf dp ra189 4.08 medium  979 aFKBD ra602 mf dp ra132 3.96 low  980 aFKBD ra602 mf dp mv 4.07 medium  981 aFKBD ra602 mf dp ra176 3.52 low  982 aFKBD ra602 mf dp ra301 3.86 low  983 aFKBD ra602 mf dp ra81 4.12 low  984 aFKBD ra602 mf dp ra350 4.10 low  985 aFKBD ra602 mf dp ra575 4.17 low  986 aFKBD ra602 mf dp ra307 3.74 low  987 aFKBD ra602 mf dp ra347 4.20 low  988 aFKBD ra602 mf dp ra554 4.17 low  989 aFKBD ra602 mf dp ra546 4.22 low  990 aFKBD ra602 mf dp ra175 4.89 low  991 aFKBD ra512 y dp ml 4.06 low  992 aFKBD ra602 y dp cha 4.00 low  993 aFKBD ra602 y dp ra84 4.52 low  994 aFKBD ra602 y dp ra206 4.73 low  995 aFKBD ra602 y dp ra209 4.12 low  996 aFKBD ra602 y dp ra80 3.91 low  997 aFKBD ra602 y dp ra549 4.16 low  998 aFKBD ra602 y dp ra189 3.68 low  999 aFKBD ra602 y dp ra132 3.53 low 1000 aFKBD ra602 y dp mv 3.70 low 1001 aFKBD ra602 y dp ra176 3.26 low 1002 aFKBD ra602 y dp ra301 3.38 low 1003 aFKBD ra602 y dp ra81 3.77 low 1004 aFKBD ra602 y dp ra350 3.83 low 1005 aFKBD ra602 y dp ra575 3.85 low 1006 aFKBD ra602 y dp ra307 3.25 low 1007 aFKBD ra602 y dp ra347 3.83 low 1008 aFKBD ra602 y dp ra554 4.09 low 1009 aFKBD ra602 y dp ra546 4.74 low 1010 aFKBD ra602 y dp ra175 4.79 low 1011 aFKBD ra602 mf ra564 ml 4.97 high 1012 aFKBD ra602 mf ra510 ml 4.85 medium 1013 aFKBD ra602 mf ra508 ml 4.49 high 1014 aFKBD ra602 mf ra557 ml 4.43 low 1015 aFKBD ra602 mf ra575 ml 4.90 low 1016 aFKBD ra602 mf ra81 ml 4.29 low 1017 aFKBD ra602 mf ra554 ml 4.79 low 1018 aFKBD ra602 mf ra546 ml 4.84 low 1019 aFKBD ra602 y ra564 ml 4.48 medium 1020 aFKBD ra602 y ra510 ml 4.26 high 1021 aFKBD ra602 y ra508 ml 4.03 high 1022 aFKBD ra602 y ra557 ml 3.93 low 1023 aFKBD ra602 y ra575 ml 4.82 medium 1024 aFKBD ra602 y ra81 ml 5.04 low 1025 aFKBD ra602 y ra554 ml 4.31 low 1026 aFKBD ra602 y ra546 ml 4.43 low 1027 aFKBD ra602 ra347 dp ml 4.41 high 1028 aFKBD ra602 ra554 dp ml 4.54 medium 1029 aFKBD ra602 ra546 dp ml 4.61 low 1030 aFKBD ra602 ra175 dp ml 5.45 low 1031 aFKBD ra602 ra307 dp ml 3.86 medium 1032 aFKBD ra602 ra522 dp ml 4.07 high 1033 aFKBD ra602 ra206 dp ml 4.12 high 1034 aFKBD ra602 ra450 dp ml 4.15 low 1035 aFKBD ra602 ra209 dp ml 4.51 medium 1036 aFKBD ra602 ra350 dp ml 4.46 low 1037 aFKBD ra602 ra176 dp ml 3.88 low 1038 aFKBD ra602 ra301 dp ml 4.03 low 1039 aFKBD ra602 ra81 dp ml 4.38 high 1040 aFKBD ra602 ra549 dp ml 4.94 medium 1041 aFKBD ra602 mv dp ml 4.44 high 1042 aFKBD ra602 ra575 dp ml 4.60 low 1043 aFKBD ra602 ra575 dp ml 4.47 low 1044 aFKBD ra301 mf dp ml 4.19 low 1045 aFKBD ra347 mf dp ml 4.63 low 1046 aFKBD ra554 mf dp ml 4.69 low 1047 aFKBD ra546 mf dp ml 4.73 low 1048 aFKBD ra175 mf dp ml 5.81 low 1049 aFKBD ra522 mf dp ml 4.18 low 1050 aFKBD ra450 mf dp ml 4.31 high 1051 aFKBD ra549 mf dp ml 5.17 low 1052 aFKBD ra176 mf dp ml 3.85 low 1053 aFKBD ra350 mf dp ml 4.67 low 1054 aFKBD ra575 mf dp ml 4.15 low 1055 aFKBD ra347 y dp ml 4.16 low 1056 aFKBD ra554 y dp ml 4.27 low 1057 aFKBD ra546 y dp ml 4.46 low 1058 aFKBD ra175 y dp ml 4.94 low 1059 aFKBD ra522 y dp ml 3.80 low 1060 aFKBD ra450 y dp ml 3.91 high 1061 aFKBD ra301 y dp ml 3.80 low 1062 aFKBD ra176 y dp ml 3.57 low 1063 aFKBD ra350 y dp ml 4.20 low 1064 aFKBD ra575 y dp ml 4.16 low 1065 aFKBD ra513 mf dp ml 4.59 high 1066 aFKBD ra602 ra559 dp ml 4.07 high 1067 aFKBD ra602 ra548 dp ml 4.02 high 1068 aFKBD ra602 ra536 dp ml 4.07 low 1069 aFKBD ra602 ra576 dp ml 3.63 high 1070 aFKBD ra602 dQ dp ml 3.33 low 1071 aFKBD ra602 ra517 dp ml 4.06 low 1072 aFKBD ra602 dN dp ml 3.32 low 1073 aFKBD ra602 N dp ml 3.35 low 1074 aFKBD ra602 Q dp ml 3.35 medium 1075 aFKBD ra602 ra560 dp ml 4.09 high 1076 aFKBD ra602 ra561 dp ml 4.13 low 1077 aFKBD ra602 ra534 dp ml 4.15 low 1078 aFKBD ra602 ra382 dp ml 3.98 low 1079 aFKBD ra602 ra531 dp ml 4.19 low 1080 aFKBD ra602 ra318 dp ml 4.06 high 1081 aFKBD ra602 ra553 dp ml 4.24 medium 1082 aFKBD ra602 ra73 dp ml 4.22 low 1083 aFKBD ra602 ra535 dp ml 4.00 low 1084 aFKBD ra602 Aca dp ml 4.42 low 1085 aFKBD ra602 ra558 dp ml 4.30 medium 1086 aFKBD ra602 ra529 dp ml 3.91 low 1087 aFKBD ra602 ra140 dp ml 3.92 low 1088 aFKBD ra348 mf dp ml 4.11 low 1089 aFKBD ra559 mf dp ml 4.25 low 1090 aFKBD ra548 mf dp ml 4.14 low 1091 aFKBD ra536 mf dp ml 4.14 low 1092 aFKBD ra576 mf dp ml 3.82 low 1093 aFKBD dQ mf dp ml 3.43 low 1094 aFKBD ra517 mf dp ml 4.18 low 1095 aFKBD dN mf dp ml 3.44 low 1096 aFKBD N mf dp ml 3.45 low 1097 aFKBD Q mf dp ml 3.46 low 1098 aFKBD ra560 mf dp ml 4.24 low 1099 aFKBD ra561 mf dp ml 4.24 low 1100 aFKBD ra534 mf dp ml 4.28 low 1101 aFKBD ra382 mf dp ml 4.10 low 1102 aFKBD ra531 mf dp ml 4.30 low 1103 aFKBD ra318 mf dp ml 4.16 low 1104 aFKBD ra553 mf dp ml 4.33 low 1105 aFKBD ra73 mf dp ml 4.32 low 1106 aFKBD ra535 mf dp ml 4.12 low 1107 aFKBD Aca mf dp ml 4.53 low 1108 aFKBD ra558 mf dp ml 4.46 low 1109 aFKBD ra529 mf dp ml 4.01 low 1110 aFKBD ra140 mf dp ml 4.04 low 1111 aFKBD ra348 y dp ml 3.77 low 1112 aFKBD ra559 y dp ml 3.88 low 1113 aFKBD ra548 y dp ml 3.80 low 1114 aFKBD ra536 y dp ml 3.78 low 1115 aFKBD ra576 y dp ml 3.45 low 1116 aFKBD dQ y dp ml 3.08 low 1117 aFKBD ra517 y dp ml 3.83 low 1118 aFKBD dN y dp ml 3.10 low 1119 aFKBD N y dp ml 3.10 low 1120 aFKBD Q y dp ml 3.12 low 1121 aFKBD ra560 y dp ml 3.91 low 1122 aFKBD ra561 y dp ml 3.88 low 1123 aFKBD ra534 y dp ml 3.94 low 1124 aFKBD ra382 y dp ml 3.77 low 1125 aFKBD ra531 y dp ml 3.98 low 1126 aFKBD ra318 y dp ml 3.88 low 1127 aFKBD ra553 y dp ml 4.01 low 1128 aFKBD ra73 y dp ml 4.00 low 1129 aFKBD ra535 y dp ml 3.77 low 1130 aFKBD Aca y dp ml 4.14 low 1131 aFKBD ra558 y dp ml 4.07 low 1132 aFKBD ra529 y dp ml 3.71 low 1133 aFKBD ra140 y dp ml 3.70 low 1134 aFKBD ra602 mf ra576 ml 4.00 low 1135 aFKBD ra602 mf ra535 ml 4.36 low 1136 aFKBD ra602 mf dN ml 3.66 low 1137 aFKBD ra602 mf dQ ml 3.68 high 1138 aFKBD ra602 mf ra536 ml 4.37 low 1139 aFKBD ra602 y ra576 ml 3.50 low 1140 aFKBD ra602 y ra535 ml 3.95 low 1141 aFKBD ra602 y dN ml 3.18 low 1142 aFKBD ra602 y dQ ml 3.23 low 1143 aFKBD ra602 y ra536 ml 3.95 low 1144 aFKBD ra602 mf dp ra559 4.06 low 1145 aFKBD ra602 mf dp ra548 4.13 low 1146 aFKBD ra602 mf dp ra517 4.14 low 1147 aFKBD ra602 mf dp N 3.46 low 1148 aFKBD ra602 mf dp Q 3.48 low 1149 aFKBD ra602 mf dp ra560 4.09 low 1150 aFKBD ra602 mf dp Aca 4.53 low 1151 aFKBD ra602 mf dp ra558 4.27 low 1152 aFKBD ra602 y dp ra559 3.66 low 1153 aFKBD ra602 y dp ra548 3.69 low 1154 aFKBD ra602 y dp ra517 3.73 low 1155 aFKBD ra602 y dp N 2.42 low 1156 aFKBD ra602 y dp Q 2.57 low 1157 aFKBD ra602 y dp ra560 3.71 low 1158 aFKBD ra602 y dp Aca 4.07 low 1159 aFKBD ra602 y dp ra558 3.91 low 1160 aFKBD ra602 mf ra545 ml 4.42 high 1161 aFKBD ra602 mf ra102 ml 4.21 medium 1162 aFKBD ra602 mf ra351 ml 4.36 low 1163 aFKBD ra602 mf aze ml 3.93 low 1164 aFKBD ra602 mf ra529 ml 4.33 low 1165 aFKBD ra602 mf ra140 ml 4.24 medium 1166 aFKBD ra602 mf ra538 ml 4.27 low 1167 aFKBD ra602 mf ra603 ml 4.15 medium 1168 aFKBD ra602 mf ra528 ml 4.06 medium 1169 aFKBD ra602 mf ra532 ml 3.88 low 1170 aFKBD ra602 mf ra539 ml 4.33 high 1171 aFKBD ra602 mf ra168 ml 4.09 low 1172 aFKBD ra602 mf ra169 ml 4.19 low 1173 aFKBD ra602 mf ra170 ml 3.96 low 1174 aFKBD ra602 mf ra542 ml 4.38 low 1175 aFKBD ra602 mf oic ml 4.19 low 1176 aFKBD ra602 mf ra524 ml 3.94 low 1177 aFKBD ra602 mf ra165 ml 4.03 medium 1178 aFKBD ra602 mf ra69 ml 4.19 low 1179 aFKBD ra602 mf ra573 ml 4.49 low 1180 aFKBD ra602 mf ra574 ml 30728.60 low 1181 aFKBD ra602 y ra545 ml 3.96 high 1182 aFKBD ra602 y ra102 ml 3.88 low 1183 aFKBD ra602 y ra351 ml 4.01 medium 1184 aFKBD ra602 y aze ml 3.48 low 1185 aFKBD ra602 y ra529 ml 3.97 low 1186 aFKBD ra602 y ra140 ml 3.89 medium 1187 aFKBD ra602 y ra538 ml 3.89 medium 1188 aFKBD ra602 y ra603 ml 3.77 high 1189 aFKBD ra602 y ra528 ml 3.67 low 1190 aFKBD ra602 y ra532 ml 3.52 low 1191 aFKBD ra602 y ra539 ml 3.98 high 1192 aFKBD ra602 y ra168 ml 3.71 medium 1193 aFKBD ra602 y ra169 ml 3.82 high 1194 aFKBD ra602 y ra170 ml 3.52 low 1195 aFKBD ra602 y ra542 ml 4.03 high 1196 aFKBD ra602 y oic ml 3.84 low 1197 aFKBD ra602 y ra524 ml 3.51 low 1198 aFKBD ra602 y ra165 ml 3.60 medium 1199 aFKBD ra602 y ra69 ml 3.82 low 1200 aFKBD ra602 y ra573 ml 4.03 low 1201 aFKBD ra602 y ra574 ml 3.87 low 1202 aFKBD ra69 mf dp ml 4.06 low 1203 aFKBD ra351 mf dp ml 4.21 low 1204 aFKBD ra102 mf dp ml 4.08 low 1205 aFKBD oic mf dp ml 4.22 low 1206 aFKBD ra542 mf dp ml 4.24 low 1207 aFKBD ra574 mf dp ml 4.21 low 1208 aFKBD ra573 mf dp ml 4.30 low 1209 aFKBD ra351 y dp ml 3.83 low 1210 aFKBD ra102 y dp ml 3.73 low 1211 aFKBD oic y dp ml 3.78 low 1212 aFKBD ra542 y dp ml 3.81 low 1213 aFKBD ra574 y dp ml 3.84 low 1214 aFKBD ra545 y dp ml 3.83 low 1215 aFKBD ra573 y dp ml 3.88 low 1216 aFKBD ra602 ra545 dp ml 4.03 low 1217 aFKBD ra602 ra351 dp ml 4.89 low 1218 aFKBD ra602 ra69 dp ml 4.10 low 1219 aFKBD ra602 ra102 dp ml 3.95 low 1220 aFKBD ra602 y dp mf 3.71 low 1221 aFKBD ra602 mf dp mf 4.07 low 1222 aFKBD ra602 mf dp ra524 3.60 low 1223 aFKBD ra540 mf dp ml 4.11 low 1224 aFKBD ra602 y dp ra562 3.72 low 1225 aFKBD ra602 mf dp ra562 4.07 low 1226 aFKBD ra602 mf dp y 3.72 low 1227 aFKBD ra602 y dp ra542 3.65 low 1228 aFKBD ra602 mf dp ra573 4.15 low 1229 aFKBD ra602 y dp ra573 3.71 low 1230 aFKBD ra602 mf dp ra574 4.03 low 1231 aFKBD ra602 rbphe dp ml 3.97 low 1232 aFKBD ra602 ra461 dp ml 3.97 low 1233 aFKBD ra602 ra462 dp ml 4.01 low 1234 aFKBD ra602 m dp ml 3.88 high 1235 aFKBD ra602 dm dp ml 3.91 low 1236 aFKBD ra602 ra458 dp ml 3.65 medium 1237 aFKBD ra602 ra459 dp ml 3.63 medium 1238 aFKBD ra602 ra456 dp ml 3.96 high 1239 aFKBD ra602 ra457 dp ml 4.03 low 1240 aFKBD ra602 ra454 dp ml 4.00 high 1241 aFKBD ra602 ra321 dp ml 4.01 low 1242 aFKBD ra602 ra452 dp ml 3.97 medium 1243 aFKBD ra602 ra306 dp ml 4.02 low 1244 aFKBD ra602 ra310 dp ml 4.18 low 1245 aFKBD ra602 ra463 dp ml 4.04 low 1246 aFKBD ra602 ra464 dp ml 3.89 low 1247 aFKBD ra602 ra466 dp ml 3.88 low 1248 aFKBD ra602 ra467 dp ml 4.01 low 1249 aFKBD ra602 ra468 dp ml 3.94 low 1250 aFKBD rbphe mf dp ml 4.02 low 1251 aFKBD ra461 mf dp ml 4.07 low 1252 aFKBD ra462 mf dp ml 4.07 low 1253 aFKBD m mf dp ml 4.00 high 1254 aFKBD dm mf dp ml 4.00 low 1255 aFKBD ra458 mf dp ml 3.75 low 1256 aFKBD ra459 mf dp ml 3.72 low 1257 aFKBD ra456 mf dp ml 4.08 low 1258 aFKBD ra457 mf dp ml 4.09 low 1259 aFKBD ra454 mf dp ml 4.10 low 1260 aFKBD ra321 mf dp ml 4.07 low 1261 aFKBD ra452 mf dp ml 4.08 low 1262 aFKBD ra306 mf dp ml 4.07 low 1263 aFKBD ra453 mf dp ml 4.16 low 1264 aFKBD ra310 mf dp ml 4.29 low 1265 aFKBD ra463 mf dp ml 4.21 low 1266 aFKBD ra464 mf dp ml 4.01 low 1267 aFKBD ra466 mf dp ml 4.01 low 1268 aFKBD ra467 mf dp ml 4.13 low 1269 aFKBD ra468 mf dp ml 4.10 low 1270 aFKBD rbphe y dp ml 3.69 low 1271 aFKBD ra461 y dp ml 3.71 low 1272 aFKBD ra462 y dp ml 3.73 low 1273 aFKBD m y dp ml 3.64 high 1274 aFKBD dm y dp ml 3.64 low 1275 aFKBD ra458 y dp ml 3.43 low 1276 aFKBD ra459 y dp ml 3.42 low 1277 aFKBD ra456 y dp ml 3.77 low 1278 aFKBD ra457 y dp ml 3.77 low 1279 aFKBD ra454 y dp ml 3.76 low 1280 aFKBD ra321 y dp ml 3.75 low 1281 aFKBD ra452 y dp ml 3.77 low 1282 aFKBD ra306 y dp ml 3.77 low 1283 aFKBD ra453 y dp ml 3.86 low 1284 aFKBD ra310 y dp ml 3.91 low 1285 aFKBD ra463 y dp ml 3.85 low 1286 aFKBD ra464 y dp ml 3.65 low 1287 aFKBD ra466 y dp ml 3.69 low 1288 aFKBD ra467 y dp ml 3.83 low 1289 aFKBD ra468 y dp ml 3.80 low 1290 aFKBD phg mf dp rbphe 3.86 low 1291 aFKBD phg mf dp ra461 3.95 low 1292 aFKBD ra602 mf dp ra462 3.97 low 1293 aFKBD ra602 mf dp m 3.96 low 1294 aFKBD ra602 mf dp ra458 3.73 low 1295 aFKBD ra602 mf dp ra456 4.12 low 1296 aFKBD ra602 mf dp ra454 4.07 low 1297 aFKBD ra602 mf dp ra452 4.06 low 1298 aFKBD ra602 mf dp ra453 4.00 high 1299 aFKBD ra602 mf dp ra310 4.32 low 1300 aFKBD ra602 mf dp ra463 3.98 low 1301 aFKBD ra602 y dp rbphe 3.54 low 1302 aFKBD ra602 y dp ra461 3.56 low 1303 aFKBD ra602 y dp ra462 3.55 low 1304 aFKBD ra602 y dp m 3.51 low 1305 aFKBD ra602 y dp ra458 3.21 low 1306 aFKBD ra602 y dp ra456 3.64 low 1307 aFKBD ra602 y dp ra454 3.64 low 1308 aFKBD ra602 y dp ra452 3.65 low 1309 aFKBD ra602 y dp ra453 3.66 low 1310 aFKBD ra602 y dp ra310 3.86 low 1311 aFKBD ra602 y dp ra463 3.66 low 1312 aFKBD ra602 mf dm ml 4.23 high 1313 aFKBD ra602 mf ra459 ml 3.92 high 1314 aFKBD ra602 mf ra457 ml 4.27 low 1315 aFKBD ra602 mf ra321 ml 4.26 low 1316 aFKBD ra602 mf ra306 ml 4.26 medium 1317 aFKBD ra602 mf ra463 ml 4.25 low 1318 aFKBD ra602 y dm ml 3.79 low 1319 aFKBD ra602 y ra459 ml 3.50 medium 1320 aFKBD ra602 y ra457 ml 3.90 low 1321 aFKBD ra602 y ra321 ml 3.90 low 1322 aFKBD ra602 y ra306 ml 3.89 low 1323 aFKBD ra602 y ra463 ml 3.91 low 1324 aFKBD ra602 ra110 dp ml 4.30 low 1325 aFKBD ra602 ra115 dp ml 4.02 medium 1326 aFKBD ra602 ra117 dp ml 4.08 high 1327 aFKBD ra602 ra116 dp ml 4.08 medium 1328 aFKBD ra602 ra113 dp ml 3.90 medium 1329 aFKBD ra602 ra114 dp ml 3.87 high 1330 aFKBD ra602 ra112 dp ml 3.85 high 1331 aFKBD ra602 ra111 dp ml 3.56 low 1332 aFKBD ra602 mf dp mi 4.13 medium 1333 aFKBD ra602 ra148 dp ml 4.13 medium 1334 aFKBD ra602 napA dp ml 4.10 medium 1335 aFKBD ra602 tic dp ml 3.95 low 1336 aFKBD ra602 ra136 dp ml 3.67 low 1337 aFKBD ra602 ra105 dp ml 3.67 low 1338 aFKBD ra602 ra137 dp ml 4.14 medium 1339 aFKBD ra602 ra101 dp ml 3.89 low 1340 aFKBD ra602 ra540 dp ml 4.04 low 1341 aFKBD ra602 ra86 dp ml 4.04 low 1342 aFKBD ra602 ra204 dp ml 4.04 low 1343 aFKBD ra602 ra134 dp ml 4.04 high 1344 aFKBD ra602 ra135 dp ml 4.20 low 1345 aFKBD ra602 ra525 dp ml 4.12 low 1346 aFKBD ra602 ra122 dp ml 4.00 medium 1347 aFKBD ra122 ra122 dp ml 4.10 low 1348 aFKBD ra122 y dp ml 3.76 low 1349 aFKBD ra110 mf dp ml 4.41 low 1350 aFKBD ra115 mf dp ml 4.14 low 1351 aFKBD ra117 mf dp ml 4.20 low 1352 aFKBD ra116 mf dp ml 4.18 low 1353 aFKBD ra113 mf dp ml 4.00 low 1354 aFKBD ra114 mf dp ml 4.00 low 1355 aFKBD ra112 mf dp ml 3.96 low 1356 aFKBD ra111 mf dp ml 3.72 low 1357 aFKBD ra109 mf dp ml 3.60 low 1358 aFKBD ra108 mf dp ml 3.55 low 1359 aFKBD ra148 mf dp ml 4.24 low 1360 aFKBD napA mf dp ml 4.24 low 1361 aFKBD ra602 mf dp ml 4.05 high 1362 aFKBD ra136 mf dp ml 3.79 low 1363 aFKBD ra105 mf dp ml 3.81 low 1364 aFKBD ra137 mf dp ml 4.27 low 1365 aFKBD ra101 mf dp ml 4.08 low 1366 aFKBD ra86 mf dp ml 4.39 low 1367 aFKBD ra134 mf dp ml 4.11 low 1368 aFKBD ra135 mf dp ml 4.26 low 1369 aFKBD ra525 mf dp ml 4.17 low 1370 aFKBD ra110 y dp ml 4.05 low 1371 aFKBD ra115 y dp ml 3.79 low 1372 aFKBD ra117 y dp ml 3.83 low 1373 aFKBD ra116 y dp ml 3.84 medium 1374 aFKBD ra113 y dp ml 3.68 low 1375 aFKBD ra114 y dp ml 3.66 low 1376 aFKBD ra112 y dp ml 3.64 low 1377 aFKBD ra111 y dp ml 3.40 low 1378 aFKBD ra109 y dp ml 3.26 low 1379 aFKBD ra108 y dp ml 3.20 low 1380 aFKBD ra148 y dp ml 3.87 low 1381 aFKBD napA y dp ml 3.88 low 1382 aFKBD ra136 y dp ml 3.50 low 1383 aFKBD ra105 y dp ml 3.43 low 1384 aFKBD ra540 y dp ml 3.77 low 1385 aFKBD ra86 y dp ml 3.74 low 1386 aFKBD ra204 y dp ml 3.70 low 1387 aFKBD ra134 y dp ml 3.76 low 1388 aFKBD ra135 y dp ml 3.94 low 1389 aFKBD ra525 y dp ml 3.86 low 1390 aFKBD ra602 mf ra540 ml 4.23 medium 1391 aFKBD ra602 y ra540 ml 3.75 low 1392 aFKBD ra602 y ra86 ml 4.16 low 1393 aFKBD ra602 mf tic ml 4.15 low 1394 aFKBD ra602 y tic ml 3.75 low 1395 aFKBD ra602 mf ra105 ml 3.95 high 1396 aFKBD ra602 y ra105 ml 3.63 high 1397 aFKBD ra602 mf ra136 ml 3.87 low 1398 aFKBD ra602 y ra136 ml 3.54 low 1399 aFKBD ra602 ra513 dp ml 5.67 high 1400 aFKBD ra602 ra120 dp ml 4.88 low 1401 aFKBD ra602 ra92 dp ml 5.10 low 1402 aFKBD ra602 ra107 dp ml 5.14 high 1403 aFKBD ra602 ra93 dp ml 5.14 medium 1404 aFKBD ra602 ra95 dp ml 5.28 low 1405 aFKBD ra602 ra96 dp ml 5.23 medium 1406 aFKBD ra602 ra87 dp ml 4.91 medium 1407 aFKBD ra602 ra104 dp ml 4.91 high 1408 aFKBD ra602 ra123 dp ml 4.90 high 1409 aFKBD ra602 ra89 dp ml 3.55 high 1410 aFKBD ra602 ra90 dp ml 3.67 medium 1411 aFKBD ra602 ra91 dp ml 4.02 medium 1412 aFKBD ra602 ra97 dp ml 5.25 low 1413 aFKBD ra602 ra94 dp ml 5.29 low 1414 aFKBD ra602 ra353 dp ml 5.43 medium 1415 aFKBD ra602 ra88 dp ml 4.80 high 1416 aFKBD ra602 ra185 dp ml 4.92 high 1417 aFKBD ra602 ra124 dp ml 4.81 high 1418 aFKBD ra602 ra526 dp ml 5.07 high 1419 aFKBD ra602 ra121 dp ml 4.86 high 1420 aFKBD ra602 ra339 dp ml 4.91 high 1421 aFKBD ra602 ra106 dp ml 4.59 high 1422 aFKBD ra602 my dp ml 4.58 high 1423 aFKBD ra602 ra133 dp ml 4.40 high 1424 aFKBD ra602 mf dp ra83 4.16 low 1425 aFKBD ra92 mf dp ml 5.26 low 1426 aFKBD ra107 mf dp ml 5.27 low 1427 aFKBD ra93 mf dp ml 5.32 low 1428 aFKBD ra95 mf dp ml 5.43 low 1429 aFKBD ra96 mf dp ml 5.44 low 1430 aFKBD Ra87 mf dp ml 5.15 low 1431 aFKBD ra602 ra108 dp ml 3.46 high 1432 aFKBD ra123 mf dp ml 5.15 low 1433 aFKBD ra89 mf dp ml 3.58 low 1434 aFKBD ra90 mf dp ml 3.66 low 1435 aFKBD ra97 mf dp ml 5.45 low 1436 aFKBD ra94 mf dp ml 5.38 low 1437 aFKBD ra353 mf dp ml 5.60 low 1438 aFKBD ra88 mf dp ml 4.94 low 1439 aFKBD ra185 mf dp ml 5.06 low 1440 aFKBD ra124 mf dp ml 5.00 low 1441 aFKBD ra526 mf dp ml 5.21 low 1442 aFKBD ra121 mf dp ml 5.02 low 1443 aFKBD ra119 mf dp ml 5.06 low 1444 aFKBD ra339 mf dp ml 5.05 low 1445 aFKBD ra106 mf dp ml 4.79 low 1446 aFKBD my mf dp ml 4.63 low 1447 aFKBD ra133 mf dp ml 4.55 low 1448 aFKBD ra513 y dp ml 4.10 high 1449 aFKBD ra120 y dp ml 4.51 high 1450 aFKBD ra92 y dp ml 4.72 low 1451 aFKBD ra107 y dp ml 4.79 low 1452 aFKBD ra93 y dp ml 4.80 low 1453 aFKBD ra95 y dp ml 4.91 low 1454 aFKBD ra96 y dp ml 4.92 low 1455 aFKBD Ra87 y dp ml 4.58 low 1456 aFKBD ra104 y dp ml 4.59 low 1457 aFKBD ra123 y dp ml 4.58 low 1458 aFKBD ra89 y dp ml 3.06 low 1459 aFKBD ra90 y dp ml 3.24 low 1460 aFKBD ra91 y dp ml 3.20 low 1461 aFKBD ra97 y dp ml 4.77 low 1462 aFKBD ra94 y dp ml 4.76 low 1463 aFKBD ra353 y dp ml 5.14 low 1464 aFKBD ra88 y dp ml 4.42 low 1465 aFKBD ra185 y dp ml 4.49 low 1466 aFKBD ra124 y dp ml 4.44 low 1467 aFKBD ra526 y dp ml 4.75 low 1468 aFKBD ra121 y dp ml 4.47 low 1469 aFKBD ra119 y dp ml 4.50 low 1470 aFKBD ra339 y dp ml 4.49 medium 1471 aFKBD ra106 y dp ml 4.25 low 1472 aFKBD my y dp ml 4.16 low 1473 aFKBD ra133 y dp ml 4.03 low 1474 raa26 ra602 mf dp ml 6.14 high 1475 raa26 ra602 y dp ml 5.89 high 1476 raa21 ra602 y dp ml 3.91 high 1477 raa21 ra602 mf dp ml 5.99 high 1478 raa7 ra602 mf dp ml 5.15 medium 1479 raa7 ra602 y dp ml 4.08 low 1480 raa6 ra602 mf dp ml 6.33 high 1481 raa6 ra602 y dp ml 6.38 high 1482 raal ra602 mf dp ml 4.47 high 1483 raal ra602 y dp ml 4.47 low 1484 raa25 ra602 mf dp ml 5.90 high 1485 raal4 ra602 mf dp ml 7.44 low 1486 raal4 ra602 y dp ml 6.60 low 1487 raal6 ra602 mf dp ml 7.30 low 1488 raal6 ra602 y dp ml 6.52 low 1489 raal2 ra602 mf dp ml 6.10 high 1490 raal2 ra602 y dp ml 5.51 high 1491 raa3 ra602 mf dp ml 5.88 low 1492 raa3 ra602 y dp ml 5.28 low 1493 aFKBD ra602 ra109 dp ml 3.50 high 1494 raal3 ra602 y dp ml 6.65 low 1495 raall ra602 mf dp ml 6.29 high 1496 raall ra602 y dp ml 4.66 high 1497 raal5 ra602 mf dp ml 5.17 low 1498 raal5 ra602 y dp ml 4.70 low 1499 raa4 ra602 mf dp ml 4.69 low 1500 raa4 ra602 y dp ml 5.39 low 1501 raa31 ra602 mf dp ml 5.00 medium 1502 raa29 ra602 mf dp ml 5.22 high 1503 raa29 ra602 y dp ml 4.59 medium 1504 raa32 ra602 mf dp ml 5.66 medium 1505 raa8 ra602 mf dp ml 4.71 high 1506 raal0 ra602 mf dp ml 4.91 high 1507 raa8 ra602 y dp ml 5.15 medium 1508 raal0 ra602 y dp ml 4.19 low 1509 raa2 ra602 mf dp ml 4.76 medium 1510 raa2 ra602 y dp ml 5.91 low 1511 raa5 ra602 mf dp ml 5.26 low 1512 raa5 ra602 y dp ml 4.60 low 1513 aFKBD ra602 ra119 dp ml 4.91 high 1514 aFKBD ra602 ra520 dp ml 4.31 high 1515 aFKBD ra602 ra569 dp ml 4.10 medium 1516 aFKBD ra602 ra570 dp ml 4.01 low 1517 aFKBD ra602 ra571 dp ml 4.01 low 1518 aFKBD ra602 ra572 dp ml 3.95 low 1519 aFKBD ra602 ra399 dp ml 4.71 low 1520 aFKBD ra602 ra515 dp ml 5.34 low 1521 aFKBD ra602 ra398 dp ml 6.89 low 1522 aFKBD ra602 y dp ml 3.65 high 1523 raa9 ra602 mf dp ml 4.02 low 1524 aFKBD ra132 mf dp ml 5.76 low 1525 aFKBD ra127 mf dp ml 5.46 high 1526 aFKBD ra126 mf dp ml 5.39 low 1527 aFKBD ra189 mf dp ml 5.91 medium 1528 aFKBD ra84 mf dp ml 5.19 high 1529 aFKBD ra83 mf dp ml 5.92 medium 1530 aFKBD ra130 mf dp ml 6.01 low 1531 aFKBD ra600 mf dp ml 5.88 high 1532 aFKBD ra565 mf dp ml 5.97 low 1533 aFKBD ra602 y dp ra83 4.44 low 1534 aFKBD tic mf dp ml 4.10 low 1535 aFKBD ra147 mf dp ml 6.18 low 1536 aFKBD ra563 mf dp ml 6.14 low 1537 aFKBD ra602 mf dp ml 5.83 low 1538 raal3 ra602 mf dp ml 7.41 low 1539 raal9 ra602 mf dp ml 5.46 low 1540 raal9 ra602 y dp ml 4.75 low 1541 raa20 ra602 mf dp ml 6.31 low 1542 raa22 ra602 ra471 dp ml 3.31 medium 1543 aFKBD ra602 ra472 dp ml 3.70 high 1544 aFKBD ra602 ra471 dp ml 5.26 high 1545 aFKBD ra602 mf ra473 ml 6.57 low 1546 aFKBD ra602 y ra473 ml 3.07 low 1547 aFKBD ra602 ra512 ra105 ml 6.45 high 1548 aFKBD ra513 ra512 ra105 ml 6.06 medium 1549 aFKBD ra513 mf ra105 ml 5.84 medium 1550 raa20 ra602 y dp ml 5.78 low 1551 aFKBD ra513 ra512 dp ml 6.23 low 1552 aFKBD ra602 ra511 dp ml 6.59 medium 1553 aFKBD ra513 ra520 dp ml 5.13 medium 1554 aFKBD ra513 ra520 ra105 ml 4.13 high 1555 raa18 ra602 mf dp ml 4.39 high 1556 rae27 ra602 mf dp ml 5.02 low 1557 raa17 ra602 mf dp ml 4.37 high 1558 afkbd phg ra500 dp ml 3.81 high 1559 afkbd phg ra501 dp ml 3.86 medium 1560 afkbd phg ra502 dp ml 3.83 low 1561 afkbd phg ra503 dp ml 3.19 low 1562 afkbd phg ra504 dp ml 3.22 low 1563 rae21 ra147 napA ra562 g 6.94 high 1564 rae29 ra147 napA ra562 g 6.67 high 1565 rae26 ra147 napA ra562 g low 1566 rae1 my df sar df medium 1567 rae10 my df sar df medium 1568 rae11 my df sar df low 1569 rae12 my df sar df low 1570 rae13 my df sar df medium 1571 rae14 my df sar df low 1572 rae16 my df sar df low 1573 rae16a my df sar df low 1574 rae17 my df sar df low 1575 rae18 my df sar df low 1576 rae19 my df sar df medium 1577 rae2 my df sar df medium 1578 rae20 my df sar df low 1579 rae21 my df sar df medium 1580 rae26 my df sar df low 1581 rae3 my df sar df medium 1582 rae4 my df sar df low 1583 rae5 my df sar df low 1584 rae9 my df sar df low 1585 afkbd phg ra655 dp ml 3.72 High 1586 afkbd phg ra656 dp ml 3.74 Med 1587 afkbd phg ra626 dp ml 3.15 Low 1588 afkbd phg ra592 dp ml 3.44 High 1589 afkbd phg ra618 dp ml 3.10 Low 1590 afkbd phg ra655 dp ml 3.72 High 1591 afkbd phg ra656 dp ml 3.74 Med 1592 afkbd phg ra626 dp ml 3.15 Low 1593 afkbd phg ra592 dp ml 3.44 High 1594 afkbd phg ra618 dp ml 3.10 Low 1595 afkbd phg ra620 dp ml 3.92 Low 1596 afkbd phg ra623 dp ml 3.96 Low 1597 afkbd ml df mi g 6.48 High 1598 aFKBD Ra602 Ra503 dp ml 5.09 high 1599 aFKBD mf dp ml 5.83 low 1600 aFKBD Ra602 mf ml 4.01 low 1601 aFKBD Ra602 y ml 3.53 low 1602 aFKBD y dp ml 3.57 low 1603 aFKBD Ra195 dp ml 4.02 low 1604 aFKBD mf dp ml 4.49 low

TABLE 8 Rapafusin compound 1605 to compound 1627. Com- Uptake, pound IC50 No. RA1 RA2 RA3 RA4 RA5 Hillslope (nM) 1605 aFKBD Gly dmPhe Pro mVal −0.9753 27.95 1606 aFKBD Ala dmPhe Pro mVal −1.164 23.73 1607 aFKBD Nva dmPhe Pro mVal −1.112 18 1608 aFKBD Leu dmPhe Pro mVal −1.105 54.14 1609 aFKBD Phe dmPhe Pro mVal −1.191 54.99 1610 aFKBD Phg dmPhe Pro mVal −0.8952 16.51 1611 eFKBD Gly dmPhe Pro mVal −1.024 48.88 1612 eFKBD Ala dmPhe Pro mVal −1.125 33.54 1613 eFKBD HoSMe dmPhe Pro mVal −0.8614 59.46 1614 aFKBD Ala dmPhe Pro mlle −0.6276 34.4 1615 aFKBD Nva dmPhe Pro mlle −0.87 12.19 1616 aFKBD Phg dmPhe Pro mlle −0.9138 100.1 1617 eFKBD Ala dmPhe Pro mlle −1.212 34.15 1618 eFKBD Nva dmPhe Pro mlle −1.195 173.1 1619 eFKBD Ala dmPhe Pro mAla −1.134 66.71 1620 eFKBD Gly dmPhe Pro mNIe −1.007 13.91 1621 eFKBD Ala dmPhe Pro mNIe −1.017 9.76 1622 eFKBD Gly dmPhe Pro mLeu −1.494 28.54 1623 eFKBD Ala dmPhe Pro mLeu −0.741 10.53 1624 aFKBD Ala dmPhe Pro mLeu −0.3876 31.45 1625 eFKBD Gly dmPhe Pro mNva −1.363 42.27 1626 eFKBD Gly dmPhe Pro dmAla −1.314 154.9 1627 eFKBD Gly dmPhe Pro Ach −1.236 261.9

In treatment, the dose of agent optionally ranges from about 0.0001 mg/kg to about 100 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.15 mg/kg to about 3 mg/kg, 0.5 mg/kg to about 2 mg/kg and about 1 mg/kg to about 2 mg/kg of the subject's body weight. In other embodiments the dose ranges from about 100 mg/kg to about 5 g/kg, about 500 mg/kg to about 2 mg/kg and about 750 mg/kg to about 1.5 g/kg of the subject's body weight. For example, depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g., 0.1-20 mg/kg) of agent is a candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage is in the range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. Unit doses can be in the range, for instance of about 5 mg to 500 mg, such as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg and 300 mg. The progress of therapy is monitored by conventional techniques and assays.

In some embodiments, an agent is administered to a human patient at an effective amount (or dose) of less than about 1 μg/kg, for instance, about 0.35 to about 0.75 μg/kg or about 0.40 to about 0.60 μg/kg. In some embodiments, the dose of an agent is about 0.35 μg/kg, or about 0.40 μg/kg, or about 0.45 μg/kg, or about 0.50 μg/kg, or about 0.55 μg/kg, or about 0.60 μg/kg, or about 0.65 μg/kg, or about 0.70 μg/kg, or about 0.75 μg/kg, or about 0.80 μg/kg, or about 0.85 μg/kg, or about 0.90 μg/kg, or about 0.95 μg/kg or about 1 μg/kg. In various embodiments, the absolute dose of an agent is about 2 μg/subject to about 45 μg/subject, or about 5 to about 40, or about 10 to about 30, or about 15 to about 25 μg/subject. In some embodiments, the absolute dose of an agent is about 20 μg, or about 30 μg, or about 40 μg.

In various embodiments, the dose of an agent may be determined by the human patient's body weight. For example, an absolute dose of an agent of about 2 μg for a pediatric human patient of about 0 to about 5 kg (e.g. about 0, or about 1, or about 2, or about 3, or about 4, or about 5 kg); or about 3 μg for a pediatric human patient of about 6 to about 8 kg (e.g. about 6, or about 7, or about 8 kg), or about 5 μg for a pediatric human patient of about 9 to about 13 kg (e.g. 9, or about 10, or about 11, or about 12, or about 13 kg); or about 8 μg for a pediatric human patient of about 14 to about 20 kg (e.g. about 14, or about 16, or about 18, or about 20 kg), or about 12 μg for a pediatric human patient of about 21 to about 30 kg (e.g. about 21, or about 23, or about 25, or about 27, or about 30 kg), or about 13 μg for a pediatric human patient of about 31 to about 33 kg (e.g. about 31, or about 32, or about 33 kg), or about 20 μg for an adult human patient of about 34 to about 50 kg (e.g. about 34, or about 36, or about 38, or about 40, or about 42, or about 44, or about 46, or about 48, or about 50 kg), or about 30 μg for an adult human patient of about 51 to about 75 kg (e.g. about 51, or about 55, or about 60, or about 65, or about 70, or about 75 kg), or about 45 μg for an adult human patient of greater than about 114 kg (e.g. about 114, or about 120, or about 130, or about 140, or about 150 kg).

In certain embodiments, an agent in accordance with the methods provided herein is administered subcutaneously (s.c.), intraveneously (i.v.), intramuscularly (i.m.), intranasally or topically. Administration of an agent described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the human patient. The dosage may be administered as a single dose or divided into multiple doses. In some embodiments, an agent is administered about 1 to about 3 times (e.g. 1, or 2 or 3 times).

The following example is provided to further illustrate the advantages and features of the present disclosure, but it is not intended to limit the scope of the disclosure. While this example is typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES

General experimental for synthesis. Syntheti reagents. Piperidine, N,N-diisopropylethylamine (DIPEA) were purchased from Alfa Aesar. Anhydrous pyridine was purchased from Acros. Solid support resin with 2-chlorotrityl chloride (Cat #: 03498) was purchased from Chem-Impex. HATU was purchased from Chemlmpex. Fmoc protected amino acid building blocks were purchased from Chemlmpex, Novabiochem or GL Biochem. Dichloromethane (DCM or CH₂Cl₂), methanol (MeOH), hexanes, ethyl acetate (EtOAc), 1,2-dichloroethane (DCE, anhydrous), N,N′-dimethylformamide (DMF, anhydrous), Hoveyda-Grubbs catalyst 2nd generation and all the other chemical reagents were purchased from Sigma-Aldrich.

Instruments for synthesis and purification. NMR spectra were recorded with Burker-400 and -500. High performance liquid chromatographic analyses were performed with Agilent LC-MS system (Agilent 1260 series, mass detector 6120 quadrupole). Orbital shaking for solid-phase reactions was performed on a Mettler-Toledo Bohdan MiniBlock system for 96 tubes (30-200 mg resin in SiliCycle tubes) or a VWR Mini Shaker (0.2-2 g resin in a plastic syringe with a fritted disc). Reagents were added with an adjustable Rainin 8-channel pipette for the MiniBlock system. Microwave reactions were performed with a Biotage Initiator Plus or Multiwave Pro with silicon carbide 24-well blocks from Anton Parr. Compound purification at 0.05-50 g scale was performed with Teledyne Isco CombiFlash Rf 200 or Biotage Isolera One systems followed by a Heidolph rotary evaporator. Purification at 1-50 mg scale was performed with Agilent HPLC system. Mixture of Rapafucins in the 45,000-compound library are purified in a high-throughput manner by SPE cartridges (Biotage, 460-0200-C, ISOLUTE, SI 2 g/6 mL) on vacuum manifold (Sigma-Aldrich, Visiprep™ SPE Vacuum Manifold, Disposable Liner, 12-port) followed by overnight drying with a custom-designed box (50 cm×50 cm×15 cm) that allows air flowing rapidly inside to remove the solvent. The high-throughput weighing of the compounds in the library was done by a Mettler-Toledo analytical balance that linked (Sartorious Entris line with RS232 port) to a computer with custom-coded electronic spreadsheet.

FKBD Example 1 4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoic acid (aFKBD)

2-allyl 1-(tert-butyl) (S)-piperidine-1,2-dicarboxylate (2)

To a solution of N-Boc homoproline 1 (6.30 g) in DMF (40 mL), Cs₂CO₃ (2.90 g) was added. The resulting suspension was stirred at RT for 5 min before the addition of allyl bromide (6.3 g). After stirring at RT for 2 h, the suspension was filtered through a pad of celite, rinsed with EtOAc (50 mL), and washed with HCl (1M, 50 mL×3). The organic layer was dried over Na₂SO₄ and co-evaporated with toluene (30 mL×2). Crude product (8.10 g) was collected as a yellow oil and was pure enough for the next step without further purification. The crude product (8.10 g) and TFA (4.3 g) were mixed well in dichloromethane (20 mL) and stirred at RT for 0.5 h. 2-allyl 1-(tert-butyl) (S)-piperidine-1,2-dicarboxylate 2 (3.00 g) was collected as a yellow oil and was pure enough for the next step without further purification.

allyl (S)-1-(4-hydroxy-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (3)

Compound 2 (3.0 g), dihydro-4,4-dimethyl-2,3-furandione (2.1 g) and DMAP (20 mg) were dissolved in toluene (20 mL) and the reaction was refluxed with an oil bath (120° C.) for 14 h. After the solvent was removed, the residue was purified by column chromatography (80-200 mesh) with EtOAc/hexane (1/3). 3 (3.50 g) was collected as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 6.04-5.80 (m, 1H), 5.36 (d, J=17 Hz, 1H), 5.31-5.25 (m, 2H), 4.68 (s, 2H), 3.76-3.56 (m, 2H), 3.50 (d, J=13 Hz, 1H), 3.40 (s, 1H), 3.20 (t, J=13 Hz, 1H), 2.37 (d, J=13 Hz, 1H), 1.84-1.61 (m, 3H), 1.61-1.34 (m, 2H), 1.24 (s, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 205.9, 170.1, 168.1, 131.4, 119.2, 69.3, 66.3, 51.6, 49.5, 44.2, 26.3, 24.8, 21.3, 21.2, 21.0.

allyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (4)

Acryloyl chloride (0.78 g) in dry CH₂Cl₂ (20 mL) was added dropwise to a mixture of compound 3 (3.50 g) and N,N-diisopropylethylamine (2.0 mL) in 50 mL CH₂Cl₂ with ice-batch over 30 min. After addition, the reaction was allowed to stir at RT for 30 min before quenched with saturated NaHCO₃ solution (20 mL). The organic phase was washed with water, dried over Na₂SO₄, concentrated and purified by column (EtOAc:Hexane=1:5) to afford product 4 (2.21 g) as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 6.39 (dd, J=17, 1.5 Hz, 1H), 6.08 (dd, J=17, 11 Hz, 1H), 5.91 (ddt, J=17, 11, 6 Hz, 1H), 5.84 (dd, J=11, 1.5 Hz, 1H), 5.35 (ddd, J=17, 2.5, 1.5 Hz, 1H), 5.28-5.25 (m, 1H), 5.26 (ddd, J=11, 2.5, 1.5 Hz, 1H), 4.66 (ddd, J=6, 4, 2.5 Hz, 2H), 4.37 (d, J=11 Hz, 1H), 4.27 (d, J=11 Hz, 1H), 3.52 (dd, J=13, 1.5 Hz, 1H), 3.23 (td, J=13, 3 Hz, 1H), 2.34 (d, J=14 Hz, 1H), 1.84-1.76 (m, 1H), 1.76-1.67 (m, 1H), 1.67-1.60 (m, 1H), 1.59-1.47 (m, 1H), 1.47-1.38 (m, 1H), 1.36 (s, 3H), 1.35 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 204.8, 169.8, 166.7, 165.5, 131.5, 131.2, 128.0, 118.9, 69.5, 69.3, 66.0, 51.3, 46.7, 43.9, 26.4, 24.9, 22.2, 21.5, 21.1. HRMS for [M+H]+ C18H25NO6, calculated: 352.1760, observed: 352.1753.

(S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylic acid (5)

compound 4 (4.2 g), Pd(PPh3)4 (230 mg), N-methylaniline (2.5 mL) were dissolved in THF (40 mL) and stirred at RT for 6 h. The reaction mixture was then diluted with EtOAc (80 mL) and washed with HCl (1M, 50 mL×3). The organic phase was separated, dried over Na2SO4, filtered and concentrated. The crude product was purified using column chromatography (200-400 mesh), where the byproduct can be eluted with 2% MeOH in dichloromethane, followed by the desired product with 3% MeOH and 0.1% AcOH in dichloromethane. 5 (2.55 g) was collected as a white solid (66%). ¹H NMR (500 MHz, CDCl₃) δ 9.96 (s, 1H), 6.39 (d, J=17 Hz, 1H), 6.08 (dd, J=17, 10 Hz, 1H), 5.85 (d, J=10 Hz, 1H), 5.30 (s, 1H), 4.55-4.30 (m, 1H), 4.32 (d, J=6 Hz, 2H), 3.53 (d, J=12 Hz, 1H), 3.24 (t, J=12 Hz, 1H), 2.35 (d, J=13 Hz, 1H), 1.91-1.60 (m, 2H), 1.60-1.42 (m, 2H), 1.36 (s, 3H), 1.34 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 204.7, 175.3, 166.8, 165.7, 131.4, 127.8, 69.6, 69.5, 51.2, 46.7, 44.0, 26.2, 24.9, 22.1, 21.8, 21.1. HRMS for [M+H]+ C15H21NO6, calculated: 312.1447, observed: 312.1444.

(E)-1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (6)

To a solution of 3,4-dimethoxybenzaldehyde (5.10 g) and 3-amino acetophenone (4.15 g) mixture in EtOH (20 mL, 95%), NaOH (0.2 g in 2 mL water) was added. The reaction mixture was stirred at RT for 6 h and a slurry of yellow precipitate was formed. The reaction mixture was then diluted with EtOAc (40 mL) and washed with water (30 mL×3). Upon concentrated, the crude product 6 (9.0 g) is pure enough for the next step.

1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propan-1-one (7)

To a solution of α,β-unsaturated ketone 6 (crude, 9.0 g) in MeOH (20 mL), Pd/C (10%, 1.61 g) was added. The reaction vessel was flushed with hydrogen gas repetitively by using a balloon of hydrogen and high vacuum. The reaction mixture was stirred at RT for 1 h before filtered through a pad of celite. Longer reaction time would render the reaction to generate undesired byproducts. The filtrate was concentrated and subject to column chromatography (50 g silica gel) and eluted with EtOAc/CH₂Cl₂/hexane (1/3/3 to 1/1/1). 7 (2.48 g) was collected as a yellow oil. ¹H NMR (500 MHz, CDCl₃) δ 7.36-7.16 (m, 3H, ar), 6.92-6.71 (m, 4H, ar), 3.86 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.81 (s, 2H, NH2), 3.23 (t, J=7.5 Hz, 2H, COCH2), 2.99 (t, J=7.4 Hz, 2H, ArCH2). ¹³C NMR (126 MHz, CDCl₃) δ 199.66 (C═O), 148.90 (ar), 147.38 (ar), 146.82 (ar), 138.03 (ar), 134.03 (ar), 129.49 (ar), 120.19 (ar), 119.61 (ar), 118.44 (ar), 113.91 (ar), 111.87 (ar), 111.35 (ar), 55.98 (OCH3), 55.87 (OCH3), 40.80 (COCH2), 29.91 (ArCH2). HRMS for [M+H]+ C17H19NO3, calculated: 286.1443, observed: 286.1436.

4-((3-(3-(3,4-dimethoxyphenyl)propanoyl)phenyl)amino)-4-oxobutanoic acid (9)

Aniline 7 (3.50 g), succinic anhydride (1.0 g) and DMAP (61 mg) were mixed in dichloromethane (30 mL). After stirring at RT for 3 h, the reaction mixture was washed with HCl (1M, 30 mL×4). Crude product (3.80 g) was collected as a white solid and was used directly in the next step without further purification. Cs₂CO₃ (1.86 g) was added into a solution of the above crude product (3.80 g) in DMF (20 mL). The resulting suspension was stirred at RT for 10 min before allyl bromide (1.50 mL) was added. The reaction mixture was stirred for an extra 2 h. The white precipitate was filtered off with a pad of celite. The filtrate was added with EtOAc (40 mL) and H₂O (40 mL). Upon stirring for 10 min, the product precipitated. Product 9 (2.11 g) was obtained by filtration, air-dried as an off-white solid, and used in the next step without further purification.

(R)-1-(3-(4-(allyloxy)-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (10)

Alcohol 9 (1.65 g) and carboxylic acid 5 (1.26 g, for synthesis see FKBD EXAMPLE 1) were dissolved in a mixture of THF (anhydrous, 5 mL) and dichloromethane (anhydrous, 10 mL). Benzoyl chloride (0.60 mL), Et3N (1.0 mL) and DMAP (18 mg) were added in order and the resulting suspension was stirred at RT for 2 h. Without further treatment, the mixture was subject to column chromatography (80-200 mesh) with EtOAc/hexane (1/2à1/1). 10 (2.50 g) was collected as a yellow foam. 1H NMR (500 MHz, CDCl₃) δ 8.08 (s, 1H), 7.65 (d, J=8 Hz, 1H), 7.46 (s, 1H), 7.28 (t, J=8 Hz, 1H), 7.01 (d, J=8 Hz, 1H), 6.77 (d, J=9 Hz, 1H), 6.69 (d, J=5 Hz, 1H), 6.67 (s, 1H), 6.39 (dd, J=17, 1.5 Hz, 1H), 6.06 (dd, J=17, 10.5 Hz, 1H), 5.90 (ddt, J=17, 10.5, 6 Hz, 1H), 5.83 (dd, J=10.5, 1.5 Hz, 1H), 5.79 (ddd, J=10.5, 8, 3.5 Hz, 1H), 5.31 (dd, J=17, 1.5 Hz, 2H), 5.31 (d, J=6 Hz, 1H), 5.22 (dd, J=10.5, 1.5 Hz, 1H), 4.60 (dt, J=6, 1.5 Hz, 2H), 4.33 (d, J=0.7 Hz, 2H), 3.86 (s, 3H), 3.85 (s, 3H), 3.46 (d, J=14 Hz, 1H), 3.09 (dd, J=18, 8 Hz, 1H), 2.78 (t, J=6 Hz, 2H), 2.70 (t, J=6 Hz, 2H), 2.62-2.48 (m, 2H), 2.36 (d, J=14 Hz, 1H), 2.30-2.16 (m, 1H), 2.13-2.00 (m, 1H), 1.74 (d, J=10.5 Hz, 2H), 1.62 (d, J=12 Hz, 1H), 1.42 (d, J=12.6 Hz, 1H), 1.36 (s, 6H). 13C NMR (126 MHz, CDCl₃) δ 205.6, 172.6, 169.8, 169.3, 166.2, 165.6, 148.9, 147.3, 140.7, 138.6, 133.5, 132.0, 131.5, 129.2, 127.8, 122.0, 120.2, 119.3, 118.4, 117.2, 111.7, 111.3, 76.5, 69.2, 65.5, 55.9, 55.9, 51.3, 46.8, 44.1, 38.1, 31.9, 31.1, 29.3, 26.1, 25.1, 22.0, 21.9, 20.9.

4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoic acid (aFKBD)

10 (2.50 g), Pd(PPh3)4 (100 mg), N-methylaniline (1.0 mL) were mixed well in THF (20 mL) at RT for 5 h. The reaction mixture was then diluted with EtOAc (50 mL) and washed with HCl (1M, 50 mL×3). The organic phase was dried over Na2SO4, filtered and concentrated. The crude product was purified by column chromatography (200-400 mesh), where the byproduct can be eluted with 2% MeOH in dichloromethane, followed by the desired product with 3% MeOH and 0.05% AcOH in dichloromethane. aFKBD (2.25 g) was collected as an off-white foam. ¹H NMR (500 MHz, CDCl₃) δ 8.40 (s, 1H), 7.62 (s, 1H), 7.48 (s, 1H), 7.26 (t, J=7.5 Hz, 1H), 7.01 (d, J=7.5 Hz, 1H), 6.97 (dd, J=16, 7 Hz, 1H), 6.86-6.74 (m, 1H), 6.74-6.58 (m, 2H), 5.85-5.68 (m, 2H), 5.39-5.24 (m, 1H), 4.29 (q, J=11 Hz, 2H), 3.86 (s, 3H), 3.84 (s, 3H), 3.46 (d, J=13 Hz, 1H), 3.13 (t, J=13 Hz, 1H), 2.74 (d, J=5.5 Hz, 2H), 2.69 (d, J=5.5 Hz, 2H), 2.63-2.48 (m, 2H), 2.36 (d, J=13 Hz, 1H), 2.30-2.15 (m, 1H), 2.15-1.99 (m, 1H), 1.85 (d, J=6 Hz, 1H), 1.75 (d, J=12 Hz, 1H), 1.63 (d, J=13 Hz, 1H), 1.55-1.38 (m, 2H), 1.34 (s, 6H). ¹³C NMR (126 MHz, CDCl₃) δ 205.6, 176.8, 170.4, 169.4, 166.4, 166.1, 148.9, 147.3, 145.9, 140.7, 138.5, 133.5, 129.2, 122.1, 121.9, 120.2, 119.5, 117.4, 111.8, 111.4, 76.6, 69.0, 55.9, 55.8, 51.4, 46.8, 44.1, 38.1, 31.6, 31.1, 29.3, 26.2, 25.0, 21.8, 20.9, 18.1. HRMS for [M+H]+ C36H44O2N11, calculated: 681.3023, observed: 681.3018.

FKBD Example 2 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (eFKBD)

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propy (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (6)

Alcohol 4 (3.8 g, 1.0 eq. For its synthesis see Liu et al. (2014) Angew. Chem. Int. Ed. 53:10049-55), carboxylic acid 5 (4.1 g, 1.2 eq. for synthesis see FKBD Example 1) and DMAP (134 mg, 0.1 eq.) were dissolved in a mixture of THF (anhydrous, 35 mL) and dichloromethane (anhydrous, 35 mL) in a round bottom 42 flask under argon protection. Et3N (4.7 mL) and benzoyl chloride (2.17 mL, 2.62 g, 1.7 eq.) were added dropwise through syringes in order and the resulting suspension was stirred at RT for 2 h. Reaction was monitored through TLC. When full conversion is achieved, the reaction mixture was diluted with 500 Ml EtOAc, washed with 5% HCl and saturated NaHCO₃. Organic phase was washed with brine and dried over Na₂SO₄. Then solvents were removed and product was purified by column chromatography (80-200 mesh) with EtOAc/hexane (1/10 to 1/3). 6 (5.3 g, 69%) was collected as a light yellow foam. ¹H NMR (500 MHz, CDCl₃) δ 7.26 (d, J=8 Hz, 1H), 6.97 (d, J=8.5 Hz, 1H), 6.93-6.89 (m, 1H), 6.86-6.81 (m, 1H), 6.78 (d, J=8.5 Hz, 1H), 6.71-6.64 (m, 2H), 6.38 (dd, J=17, 1.5 Hz, 1H), 6.06 (dd, J=17, 10.5 Hz, 1H), 5.82 (dd, J=10.5, 1.5 Hz, 1H), 5.78 (dd, J=8, 6 Hz, 1H), 5.29 (d, J=5 Hz, 1H), 4.53 (s, 2H), 4.36 (d, J=11 Hz, 1H), 4.27 (d, J=11 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.48 (d, J=13 Hz, 1H), 3.17 (td, J=13, 3.0 Hz, 1H), 2.67-2.44 (m, 2H), 2.37 (d, J=14 Hz, 1H), 2.32-2.18 (m, 1H), 2.14-1.99 (m, 1H), 1.83-1.65 (m, 2H), 1.65-1.56 (m, 1H), 1.50-1.43 (m, 2H), 1.48 (s, 9H), 1.35 (s, 3H), 1.35 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 204.8, 169.4, 167.8, 166.4, 165.4, 158.1, 148.9, 147.3, 141.3, 133.4, 131.2, 129.7, 127.9, 120.2, 119.8, 114.2, 113.2, 111.7, 111.3, 82.3, 76.7, 69.2, 65.7, 55.9, 55.8, 51.4, 46.6, 44.0, 37.9, 31.2, 28.0, 26.4, 25.0, 22.1, 21.6, 21.1. HRMS for [M+H]+ C38H49NO11, calculated: 696.3384, observed: 696.3386.

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (eFKBD)

Compound 6 (5.3 g, 1.0 eq.) was dissolved in 60 mL of dichloromethane in a round-bottom flask under Ar protection. Then TFA (17 mL, 11.4 g, 13 eq.) was added through a syringe in 3 portions during 3.5 h while stirring at room temperature. The reaction was monitored through TLC. When full conversion was achieved, solvents and TFA were removed under vacuum. Product was purified by column chromatography (80-200 mesh) with EtOAc/hexane (1/5à1/1). eFKBD (4.6 g, 96%) was collected as a light yellow foam. ¹H NMR (500 MHz, CDCl₃) δ 7.28 (dd, J=3.5 Hz, 3.5 Hz, 1H), 6.88 (d, J=8.5 Hz, 1H), 6.83-6.81 (m, 2H), 6.80-6.78 (m, 1H), 6.69-6.67 (m, 2H), 6.37 (d, J=8.5 Hz, 1H), 6.05-6.02 (m, 1H), 5.83-5.72 (m, 2H), 5.30-5.28 (dd, J=10, 5 Hz, 1H), 4.67 (dd, J=10, 5 Hz, 1H), 4.17 (dd, J=10, 6 Hz, 2H), 3.48-3.45 (m, 1H), 3.24-3.22 (m, 1H), 2.61-2.55 (m, 2H), 2.38 (m, 1H), 2.23 (m, 1H), 2.04 (m, 1H), 1.79 (m, 1H), 1.62 (m, 1H), 1.33 (m, 1H), 1.30 (m, 1H), 1.25 (s, 3H), 1.24 (s, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 204.6, 169.2, 166.7, 165.7, 157.9, 149.0, 147.5, 141.7, 131.4, 129.9, 127.9, 120.0, 115.4, 111.8, 111.4, 111.1, 69.3, 65.2, 60.5, 55.9, 51.7, 44.1, 38.0, 31.4, 22.1, 21.1, 14.2. HRMS for [M+H]+ C34H42NO11, calculated: 640.2758, observed: 640.2761.

FKBD Example 3 4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-morpholinopropyl)phenylamino)-4-oxobutanoic acid (Raa1)

1-(3-nitrophenyl)prop-2-en-1-one (2)

Paraformaldehyde (36 g, 120 mmol) was added to a stirred solution of 1-(3-nitrophenyl)ethanone 1 (20 g, 120 mmol), N-methylanilinium trifluoroacetate (26.8 g, 120 mmol) and TFA (1.4 g, 12 mmol) in THF (300 mL) at rt, the resultant reaction was heated to reflux for 16 h. The solvent was removed in vacuo, the residue was diluted with water (100 mL) and EA (200 mL). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to afford compound 2 as a yellow solid (14.2 g, crude) used for next step directly without purification. [M+H]⁺=178.1.

3-morpholino-1-(3-nitrophenyl)propan-1-one (3)

To a solution of 2 (12 g, 33.9 mmol, crude) in DMF (30 mL) was added Morpholine (2.95 g, 33.9 mmol), followed by 4-methylbenzenesulfonic acid (5.83 g, 33.9 mmol). After stirring at room temperature for 5 h, quenched the reaction with H₂O (50 mL), extracted with EA (100 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-10% as eluent) to afford compound 3 (6.6 g, 74%) as a yellow oil. [M+H]⁺=265.2

1-(3-aminophenyl)-3-morpholinopropan-1-one (4)

To a solution of 3 (4.2 g, 15.9 mmol) in THF (20 mL) was added 10% Pd/C (wet, 840 mg) at rt. The resulting reaction mixture was hydrogenated with H₂ (g) at rt for 8 h. The reaction mixture was then filtered and concentrated in vacuo to afford crude compound 4 (3.46 g, crude) as a yellow oil used for next step directly. [M+H]⁺=235.1

tert-butyl 4-(3-(3-morpholinopropanoyl)phenylamino)-4-oxobutanoate (6)

To a solution of 4 (5.05 g, 21.5 mmol) and 4-tert-butoxy-4-oxobutanoic acid 5 (4.86 g, 27.95 mmol) in DMF (20 mL) was added DIPEA (5.55 g, 43 mmol) followed by HATU (10.62 g, 27.95 mmol) at rt. The resulting reaction mixture was stirred at rt for 2 h. Quenched the reaction with H₂O (50 mL), extracted with EA (100 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 6 (4.3 g, 51%) as a yellow solid. [M+H]⁺=391.0

(R)-tert-butyl 4-(3-(1-hydroxy-3-morpholinopropyl)phenylamino)-4 oxobutanoate (7)

To a solution of ketone 6 (4.1 g, 10.5 mmol) in anhydrous THF (40 mL) was added (+) DIPChloride (42 mmol) in heptane (1.7 M, 24.7 mL) at −20° C. The resulting reaction mixture was stirred at −20° C. until complete conversion of 6, the quenched with 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (7 g, 47.25 mmol) by forming an insoluble complex. After stirring at rt for another 30 min, the suspension was filtered through a pad of celite and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 7 (1.0 g, 24%) as an off white solid. [M+H]⁺=393.0

(S)—((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-morpholinopropyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (1.0 g, 2.55 mmol) and 8 (952 mg, 3.06 mmol) in anhydrous DCM (25 mL) was cooled to -20° C. before a solution of DCC (630 mg, 3.06 mmol) in anhydrous DCM (2 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 31 mg, 0.255 mmol) under argon atmosphere. The resulting white suspension was stirred at −20° C. for 2 h. The reaction mixture was then filtered and the filtrate were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/DCM=0-5% as eluent) to afford compound 9 (1.3 g, 76%) as a white solid. [M+H]⁺=686.0

4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-morpholinopropyl)phenylamino)-4-oxobutanoic acid (RAa-1)

To a solution of 9 (1.3 g, 1.9 mmol) in DCM (10 mL) was added TFA (2 mL) at rt. The resulting mixture was stirred at rt for 3 h. The reaction mixture was charged to silica-gel flash column directly (CH₃OH/DCM=0-5% as eluent) to afford RAa-1 as a white solid (620 mg, 51%).

FKBD Example 4 4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-4-morpholinobutyl)phenylamino)-4-oxobutanoic acid (Raa2)

3-(3-nitrobenzoyl)-dihydrofuran-2(3H)-one (3)

To a stirred solution of dihydrofuran-2(3H)-one 2 (6.02 g, 70 mmol) in anhydrous THF (60 mL) was added LiHMDS (1M in THF, 77 mL, 77 mmol) at −78° C. and stirred for 2 h under argon atmosphere. Then the solution of 3-nitrobenzoyl chloride 1 (6.5 g, 35 mmol) in anhydrous THF (10 mL) was added at −78° C. The resultant reaction mixture was slowly warmed to rt and stirred at rt for 16 h. Quenched the reaction with saturated NH₄Cl_(aq) (20 mL), extracted with EA (100 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to afford compound 3 (8.5 g, crude) as a yellow oil used for next step directly without purification. [M+H]⁺=236.1

4-bromo-1-(3-nitrophenyl)butan-1-one (4)

A solution of 3 (25.9 g, 110 mmol, crude) in 40% HBr (150 mL) was heated to 70° C. for 2 h. The reaction mixture was cooled to rt and adjusted the pH to 5-6 with saturated NaHCO_(3aq), extracted with EA (200 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, EA/PE=0-10% as eluent) to afford compound 4 (18.5 g, 74% for 2 steps) as a yellow oil.

4-morpholino-1-(3-nitrophenyl)butan-1-one (5)

To a solution of 4 (8.5 g, 31.25 mmol) and Morpholine (2.72 g, 31.25 mmol) in CH₃CN (100 mL) was added K₂CO₃ (8.64 g, 62.5 mmol) at rt. The resulting reaction mixture was heated to reflux for 2 h. The reaction mixture was then filtered and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 5 (4.6 g, 53%) as a yellow oil. [M+H]⁺=279.2

1-(3-aminophenyl)-4-morpholinobutan-1-one (6)

A solution of 5 (5.9 g, 21.2 mmol) in THF (60 mL) was added 10% Pd/C (wet, 1.18 g) at rt. The resulting reaction mixture was hydrogenated with H₂ (g) at rt for 10 h. The reaction mixture was then filtered and concentrated in vacuo to afford crude compound 6 (4.8 g, crude) as a yellow solid used for next step directly. [M+H]⁺=249.0

To a solution of 6 (4.8 g, 19.35 mmol) and 4-tert-butoxy-4-oxobutanoic acid 7 (4.86 g, 27.95 mmol) in DMF (15 mL) was added DIPEA (5.0 g, 38.7 mmol) followed by HATU (9.56 g, 25.15 mmol) at rt. The resulting reaction mixture was stirred at rt for 2 h. Quenched the reaction with H₂O (50 mL), extracted with EA (100 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 8 (6.6 g, 84%) as a yellow solid. [M+H]⁺=405.0

(R)-tert-butyl 4-(3-(1-hydroxy-4-morpholinobutyl)phenylamino)-4-oxobutanoate (9)

To a solution of ketone 8 (5.0 g, 12.4 mmol) in anhydrous THF (20 mL) was added (+) DIPChloride (49.6 mmol) in heptane (1.7 M, 29 mL) at −20° C. The resulting reaction mixture was stirred at −20° C. until complete conversion of 8, then quenched with 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (8.3 g, 55.8 mmol) by forming an insoluble complex. After stirring at rt for another 30 min, the suspension was filtered through a pad of celite and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 9 as an off white solid (2.5 g, 50%). [M+H]⁺=407.3

(S)—((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-4-morpholinobutyl)1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (11)

A solution of 9 (2.45 g, 6.05 mmol) and 10 (2.29 g, 7.38 mmol) in anhydrous DCM (40 mL) was cooled to −20° C. before a solution of DCC (1.52 g, 7.38 mmol) in anhydrous DCM (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 75 mg, 0.615 mmol) in anhydrous DCM (1 mL) under argon atmosphere. The resulting white suspension was stirred at −20° C. for 2 h. The reaction mixture was then filtered and the filtrate were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/DCM=0-5% as eluent) to afford compound 11 as a white solid (3 g, 69%). [M+H]⁺=700.0

4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-4-morpholinobutyl)phenylamino)-4-oxobutanoic acid (Raa2)

To a solution of 11 (1.0 g, 1.42 mmol) in DCM (10 mL) was added TFA (2 mL) at rt. The resulting mixture was stirred at rt for 2 h. The reaction mixture was charged to silica-gel flash column directly (CH₃OH/DCM=0-5% as eluent) to afford Raa2 (550 mg, 60%) as a white solid.

FKBD Example 5 4-(3-((R)-3-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)propyl)phenylamino)-4-oxobutanoic acid (Raa3)

tert-butyl 4-(3-(3-nitrophenyl)-3-oxopropyl)piperazine-1-carboxylate (3)

To a solution of 1 (10 g, 28.2 mmol, crude) in DMF (20 mL) was added DIPEA (3.64 g, 28.2 mmol), followed by 2 (5.24 g, 28.2 mmol). After stirring at room temperature for 2 h, quenched the reaction with H₂O (100 mL), extracted with EA (100 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-10% as eluent) to afford compound 3 as a yellow oil (6.1 g, 60%). [M+H]⁺=364.2

tert-butyl 4-(3-(3-aminophenyl)-3-oxopropyl)piperazine-1-carboxylate (4)

A solution of 3 (6.1 g, 15.9 mmol) in THF (50 mL) was added 10% Pd/C (wet, 1.22 g) at rt. The resulting reaction mixture was hydrogenated with H₂ (g) at rt for 8 h. The reaction mixture was then filtered and concentrated in vacuo to afford crude compound 4 as a brown solid (5.5 g, crude) used for next step directly. [M+H]⁺=334.3

tert-butyl 4-(3-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-oxopropyl)piperazine-1-carboxylate (6)

To a solution of 4 (5.2 g, 15.6 mmol) and 4-tert-butoxy-4-oxobutanoic acid 5 (3.53 g, 20.27 mmol) in DMF (35 mL) was added DIPEA (5.04 g, 38.99 mmol) followed by HATU (7.71 g, 20.27 mmol) at rt. The resulting reaction mixture was stirred at rt for 4 h. Quenched the reaction with H₂O (50 mL), extracted with EA (100 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, PE/EA=0-50% as eluent) to afford compound 6 (4.3 g, 56%) as a yellow solid. [M+H]⁺=490.4

(R)-tert-butyl 4-(3-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-hydroxypropyl)piperazine-1-carboxylate (7)

To a solution of ketone 6 (3.8 g, 7.76 mmol) in anhydrous THF (30 mL) was added (+) DIPChloride (38.8 mmol) in heptane (1.7 M, 23 mL) at −20° C. The resulting reaction mixture was stirred at −20° C. until complete conversion of 6, the quenched with 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (6.32 g, 42.68 mmol) by forming an insoluble complex. After stirring at rt for another 30 min, the suspension was filtered through a pad of celite and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 7 as an off white solid (1.9 g, 51%). [M+H]⁺=492.3

tert-butyl 4-((R)-3-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)propyl)piperazine-1-carboxylate (9)

A solution of 7 (1.03 g, 2.1 mmol) and 8 (784 mg, 2.52 mmol) in anhydrous DCM (20 mL) was cooled to −20° C. before a solution of DCC (865 mg, 4.2 mmol) in anhydrous DCM (2 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 26 mg, 0.21 mmol) under argon atmosphere. The resulting white suspension was stirred at −20° C. for 2 h. The reaction mixture was then filtered and the filtrate were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/DCM=0-5% as eluent) to afford compound 9 as a yellow solid (1.2 g, 72%). [M+H]⁺=784.9

4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(piperazin-1-yl)propyl)phenylamino)-4-oxobutanoic acid (10)

To a solution of 9 (1.2 g, 1.9 mmol) in DCM (6 mL) was added TFA (3 mL) at rt. The resulting mixture was stirred at rt for 3 h. The reaction mixture was concentrated in vacuo to afford compound 10 (1.1 g, crude) as a yellow solid. [M+H]⁺=628.9

4-(3-((R)-3-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)propyl)phenylamino)-4-oxobutanoic acid (Raa3)

To a solution of 10 (1.1 g, 1.74 mmol) in DMF (4 mL) was added Na₂CO₃ (369 mg, 3.48 mmol) followed by FmocChloride (450 mg, 1.74 mmol) at rt. The resulting reaction mixture was stirred at rt for 30 min. Quenched the reaction with H₂O (10 mL), extracted with EA (30 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford Raa3 (680 mg, 46%) as a white solid.

FKBD Example 6 4-(3-((R)-4-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)butyl)phenylamino)-4-oxobutanoic acid (Raa4)

tert-butyl 4-(4-(3-nitrophenyl)-4-oxobutyl)piperazine-1-carboxylate (3)

To a solution of 1 (10.5 g, 38.6 mmol) and 2 (7.2 g, 38.6 mmol) in CH₃CN (100 mL) was added K₂CO₃ (10.7 g, 77.2 mmol) at rt. The resulting reaction mixture was heated to reflux for 2 h. The reaction mixture was then filtered and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 3 (8.3 g, 57%) as a yellow solid. [M+H]⁺=378.0

tert-butyl 4-(4-(3-aminophenyl)-4-oxobutyl)piperazine-1-carboxylate (4)

A solution of 3 (8.3 g, 22 mmol) in THF (60 mL) was added 10% Pd/C (wet, 1.66 g) at rt. The resulting reaction mixture was hydrogenated with H₂ (g) at rt for 10 h. The reaction mixture was then filtered and concentrated in vacuo to afford crude compound 4 (7.4 g, crude) as a yellow solid used for next step directly. [M+H]⁺=348.3

tert-butyl 4-(3-(4-morpholinobutanoyl)phenylamino)-4-oxobutanoate (6)

To a solution of 4 (7.4 g, 21.3 mmol) and 4-tert-butoxy-4-oxobutanoic acid 5 (4.82 g, 27.6 mmol) in DMF (15 mL) was added DIPEA (5.5 g, 42.6 mmol) followed by HATU (10.5 g, 27.69 mmol) at rt. The resulting reaction mixture was stirred at rt for 2 h. Quenched the reaction with H₂O (50 mL), extracted with EA (100 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 6 (8.5 g, 79%) as a yellow solid. [M+H]⁺=504.0

(R)-tert-butyl 4-(4-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-4-hydroxybutyl)piperazine-1-carboxylate (7)

To a solution of ketone 6 (4.5 g, 8.9 mmol) in anhydrous THF (20 mL) was added (+) DIPChloride (35.6 mmol) in heptane (1.7 M, 21 mL) at −20° C. The resulting reaction mixture was stirred at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (5.9 g, 40.0 mmol) by forming an insoluble complex. After stirring at rt for another 30 min, the suspension was filtered through a pad of celite and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/EA=0-5% as eluent) to afford compound 7 as an off white solid (2.5 g, 55%). [M+H]⁺=506.0

tert-butyl 4-((R)-4-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-4-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)butyl)piperazine-1-carboxylate (9)

A solution of 7 (2.3 g, 4.5 mmol) and 8 (1.68 g, 5.4 mmol) in anhydrous DCM (30 mL) was cooled to −20° C. before a solution of DCC (1.1 g, 5.4 mmol) in anhydrous DCM (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 55 mg, 0.615 mmol) in anhydrous DCM (1 mL) under argon atmosphere. The resulting white suspension was stirred at −20° C. for 2 h. The reaction mixture was then filtered and the filtrate were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 9 as a white solid (2.9 g, 80%). [M+H]⁺=799.5

4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-4-(piperazin-1-yl)butyl)phenylamino)-4-oxobutanoic acid (10)

To a solution of 9 (2.9 g, 3.6 mmol) in DCM (10 mL) was added TFA (3 mL) at rt. The resulting mixture was stirred at rt for 4 h. The reaction mixture was charged to silica-gel flash column directly (CH₃OH/DCM=0-5% as eluent) to afford compound 10 (2.6 g, crude) as a yellow solid used for next step directly. [M+H]⁺=643.4

4-(3-((R)-4-(4-(((9H-fluoren-9-yl)methoxy)carbonyl)piperazin-1-yl)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)butyl)phenylamino)-4-oxobutanoic acid (Raa4)

To a solution of 10 (1.2 g, 1.62 mmol) in DMF (2 mL) was added Na₂CO₃ (343 mg, 3.24 mmol) followed by FmocChloride (419 mg, 1.62 mmol) at rt. The resulting reaction mixture was stirred at rt for 30 min. Quenched the reaction with H₂O (10 mL), extracted with EA (30 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford Raa4 (570 mg, 40%) as a white solid.

FKBD Example 7 4-(5-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-ylamino)-4-oxobutanoic acid (Raa5)

1-(5-aminopyridin-3-yl)ethanone (2)

To a solution of 1 (13 g, 78.3 mmol) in THF (100 mL) was added 10% Pd/C (wet, 8.0 g) at rt. The resulting reaction mixture was stirred at rt for 10 h under H₂ (g). The reaction mixture was then filtered and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 2 (10 g, 94%) as a yellow solid. [M+H]⁺=137.0

(E)-1-(5-aminopyridin-3-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (4)

To a solution of 2 (6.5 g, 47.8 mmol) and 3 (7.9 g, 47.8 mmol) in CH₃OH (60 mL) was added LiOH.H₂O (2 g, 47.8 mmol) at 0° C. The resulting reaction mixture was stirred at rt for 3 h. The solvent was removed in vacuo and the residue was diluted with DCM and H₂O. The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 4 (1.8 g, 13%) as a yellow solid. [M+H]⁺=285.0

(E)-tert-butyl 4-(5-(3-(3,4-dimethoxyphenyl)acryloyl)pyridin-3-ylamino)-4-oxobutanoate (6)

To a solution of 4 (1.8 g, 6.3 mmol) and 4-tert-butoxy-4-oxobutanoic acid 5 (1.1 g, 6.3 mmol) in DCM (35 mL) was added Et₃N (12.7 g, 12.6 mmol) followed by T₃P (50% in EtOAc, 8.0 g, 12.6 mmol) at rt. The resulting reaction mixture was stirred at rt for 1 h. Quenched the reaction with H₂O (20 mL), extracted with DCM (40 mL×2). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 6 (1.88 g, 68%) as a yellow solid. [M+H]⁺=440.9

tert-butyl 4-(5-(3-(3,4-dimethoxyphenyl)propanoyl)pyridin-3-ylamino)-4-oxobutanoate (7)

A solution of 6 (1.88 g, 4.27 mmol) in THF (50 mL) and Methanol (5 mL) was added 10% Pd/C (wet, 380 mg) at rt. The resulting reaction mixture was hydrogenated with H₂ (g) at rt for 4 h. The reaction mixture was then filtered and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 7 (1.34 g, 71%) as a brown solid. [M+H]⁺=442.9

(R)-tert-butyl 4-(5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)pyridin-3-ylamino)-4-oxobutanoate (8)

To a solution of ketone 7 (1.34 g, 3.0 mmol) in anhydrous THF (20 mL) was added (+) DIPChloride (12.0 mmol) in heptane (1.7 M, 7.05 mL) at −20° C. The resulting reaction mixture was stirred at −20° C. until complete conversion of 7, then quenched with 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (2.0 g, 13.5 mmol) by forming an insoluble complex. After stirring at rt for another 30 min, the suspension was filtered through a pad of celite and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 8 (0.99 g, 74%) as a white solid. [M+H]⁺=445.0

(S)—((R)-1-(5-(4-tert-butoxy-4-oxobutanamido)pyridin-3-yl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (10)

A solution of 8 (990 mg, 2.22 mmol) and 9 (827 mg, 2.66 mmol) in anhydrous DCM (20 mL) was cooled to −20° C. before a solution of DCC (548 mg, 2.66 mmol) in anhydrous DCM (2 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 27 mg, 0.22 mmol) in anhydrous DCM (1 mL) under argon atmosphere. The resulting white suspension was stirred at −20° C. for 2 h. The reaction mixture was then filtered and the filtrate were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/DCM=0-5% as eluent) to afford compound 10 (1.3 g, 79%) as a white solid. [M+H]⁺=738.0

4-(5-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-ylamino)-4-oxobutanoic acid (Raa5)

To a solution of 10 (1.3 g, 1.76 mmol) in DCM (10 mL) was added TFA (5 mL) at rt. The resulting mixture was stirred at rt for 2 h. The reaction mixture was charged to silica-gel flash column directly (CH₃OH/DCM=0-5% as eluent) to afford Raa5 (960 mg, 80%) as a white solid.

FKBD Example 8 4-(6-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-2-ylamino)-4-oxobutanoic acid (Raa6)

(E)-1-(6-aminopyridin-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (3)

To a solution of 1 (3.75 g, 27.57 mmol) and 2 (4.58 g, 27.57 mmol) in CH₃OH (40 mL) was added LiOH.H₂O (1.74 g, 41.35 mmol) at rt. The resulting reaction mixture was stirred at rt for 3 h. The solvent was removed in vacuo and the residue was diluted with DCM and H₂O. The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 3 (3.2 g, 41%) as a yellow solid. [M+H]⁺=285.0

(E)-tert-butyl 4-(6-(3-(3,4-dimethoxyphenyl)acryloyl)pyridin-2-ylamino)-4-oxobutanoate (5)

To a solution of 3 (3.2 g, 11.26 mmol) and 4-tert-butoxy-4-oxobutanoic acid 4 (2.35 g, 13.5 mmol) in Pyridine (10 mL) was added POCl₃ (2.58 g, 16.89 mmol) at 0° C. The resulting reaction mixture was stirred at 0° C. for 15 min. Quenched the reaction with H₂O (20 mL), extracted with EA (30 mL×3). The organic extracts were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 5 (2.45 g, 49%) as a yellow solid. [M+H]⁺=440.9

tert-butyl 4-(6-(3-(3,4-dimethoxyphenyl)propanoyl)pyridin-2-ylamino)-4-oxobutanoate (6)

A solution of 5 (2.45 g, 5.56 mmol) in THF (30 mL) was added 10% Pd/C (wet, 500 mg) at rt. The resulting reaction mixture was hydrogenated with H₂ (g) at rt for 4 h. The reaction mixture was then filtered and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, Methanol/DCM=0-5% as eluent) to afford compound 6 (1.5 g, 61%) as a yellow solid. [M+H]⁺=443.3

(R)-tert-butyl 4-(6-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)pyridin-2-ylamino)-4-oxobutanoate (7)

To a solution of ketone 6 (1.4 g, 3.16 mmol) in anhydrous DCM (20 mL) was added (+) DIPChloride (12.64 mmol) in heptane (1.7 M, 7.5 mL) at −20° C. The resulting reaction mixture was stirred at −20° C. until complete conversion of 7, then quenched with 2,2′-(ethane-1,2-diylbis(oxy))diethanamine (2.1 g, 14.22 mmol) by forming an insoluble complex. After stirring at rt for another 30 min, the suspension was filtered through a pad of celite and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/EA=0-5% as eluent) to afford compound 7 (1.0 g, 71%) as a white solid. [M+H]⁺=445.3

(S)—((R)-1-(6-(4-tert-butoxy-4-oxobutanamido)pyridin-2-yl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (1.0 g, 2.24 mmol) and 8 (836 mg, 2.69 mmol) in anhydrous DCM (20 mL) was cooled to −20° C. before a solution of DCC (554 mg, 2.69 mmol) in anhydrous DCM (2 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 27 mg, 0.22 mmol) in anhydrous DCM (1 mL) under argon atmosphere. The resulting white suspension was stirred at −20° C. for 2 h. The reaction mixture was then filtered and the filtrate were dried over Na₂SO₄ and concentrated in vacuo to give a crude product which was further purified by column (SiO₂, CH₃OH/DCM=0-5% as eluent) to afford compound 9 (0.38 g, 23%) as a white solid. [M+H]⁺=738.4

4-(6-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-2-ylamino)-4-oxobutanoic acid (Raa6)

To a solution of 9 (0.38 g, 1.76 mmol) in DCM (5 mL) was added TFA (2 mL) at rt. The resulting mixture was stirred at rt for 2 h. The reaction mixture was charged to silica-gel flash column directly (CH₃OH/DCM=0-5% as eluent) to afford Raa6 (310 mg, 89%) as a white solid.

FKBD Example 9 4-((6-((R)—(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyrazin-2-yl)amino)-4-oxobutanoic acid (Raa7)

6-(1-butoxyvinyl)pyrazin-2-amine (3)

To a solution of 1 (16 g, 124 mmol) in ethylene glycol (150 mL) was added Pd(AcO)₂ (0.8 g, 3.7 mmol) and DPPF (4.12 g, 7.4 mmol) at rt. Degassed by Ar₂, and then 2 and Et₃N was injected sequentially. The reaction mixture was heated to reflux and reacted for 1.5 h. The product mixture was poured into water (300 ml), extracted with DCM (100 ml*3). Combined the organic phase and washed with brine (100 ml*3). Filtered and concentrated to get 3 (12 g, 50%) as white solid. [M+H]⁺=194

1-(6-aminopyrazin-2-yl)ethan-1-one (4)

To a solution of 3 (12 g, 62 mmol) in DCM (50 ml) was added 5% HCl (20 ml). The reaction mixture was stirred at rt for 0.5 h. Poured the product mixture into water (200 ml), adjusted pH to 8-9 with K₂CO₃ (aq). Extracted with DCM (50 ml*6), combined the organic phase and concentrated to get the crude. Purified by silica gel chromatography (PE/EA=20-30% as eluent) to give product 4 (2.9 g, 34%) as yellow solid. [M+H]⁺=138

(E)-1-(5-amiopyrazin-2-yl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (6)

To a solution of 4 (2.9 g, 21 mmol) in MeOH (20 ml) was added LiOH (1.74 g, 42 mol) and 5 (3.43 g, 21 mmol). The reaction mixture was stirred at 40° C. for 1 h. Poured the product mixture into water (200 ml), filtered until no more precipitation, washed the solid cake with water, and then little MeOH. Dried to get product 6 (3.8 g, 64.5%) as yellow solid. [M+H]⁺=286

tert-butyl(E)-4-((6-(3-(3,4-dimethoxyphenyl)acryloyl)pyrazin-2-yl)amino-4-oxobutanoate (8)

To a solution of 8 (3.8 g, 133 mmol) and 7 (4.64 g, 266 mmol) in pyridine (100 ml) was added POCl₃(6.12 g, 400 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 30 min. Poured the product mixture into water (300 ml), extracted with DCM (100 ml*3), combined the organic phase and washed with brine (100 ml*5). Dried over Na₂SO₄, filtered and concentrated to get the crude. Purified by silica gel chromatography (MeOH/DCM=1-2% as eluent) to give product 8 (5 g, 68%) as yellow solid. [M+H]⁺=442

tert-butyl 4-((6-(3-(3,4-dimethoxyphenyl)propannoyl)pyrazin-2-yl)amino)-4-oxobutanoate (9)

To a solution of 8 (5.0 g, 113 mmol) in THF was added Pd/C (500 mg, 10%), the reaction mixture was degassed with H₂*5, stirred at rt for 4 h. Filtered and concentrated the filtrate to get the crude. Purified by silica gel chromatography (MeOH/DCM=1-2% as eluent) to give product 9 (2.0 g, 40%) as yellow solid. [M+H]⁺=444

tert-butyl (R)4-((6-(3-(3,4-dimethoxyphenyl)-1-hydroxyphenyl)pyrazin-2-yl)amino)-4-oxobutanoate (11)

To a solution of 9 (2.0 g, 45 mmol) in DCM (50 ml) was added DIPCl (14.5 g, 450 mmol) at −20° C., degassed with Ar₂. The reaction mixture was stirred at −20° C. for 5 h. Quenched with 10 (6.75 g, 455 mmol). The product mixture was concentrated directly, and the brown residue was purified by silica gel chromatography (MeOH/DCM=2-5% as eluent) to give product 11 (1.0 g, 50%) as yellow solid. [M+H]⁺=446

(R)-1-(6-(4-tert-butoxy)-4-oxobutanamido)pyrazin-2-yl)-3-(3,4-dimethoxyphenyl(S)-1(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (13)

To a solution of 11 (1.0 g, 22 mmol) in DCM (30 ml) was added 12 (1.05 g, 34 mmol) at −20° C., and degassed with Ar₂, then DCC (0.7 g, 34 mmol) and DMAP (0.03 g, 2.2 mmol) in DCM was injected sequentially. The reaction mixture was stirred at −20° C. for 1 h. Filtered and washed the solid cake with DCM (20 ml), the filtrate was combined and evaporated to get the crude. Purified by silica gel chromatography (MeOH/DCM=1-2% as eluent) to give product 13 (1.8 g, 85%) as yellow solid. [M+H]⁺=739

4-((6-((R)—(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyrazin-2-yl)amino)-4-oxobutanoic acid (Raa7)

To a solution of 13 (1.8 g, 24 mmol) in DCM (20 ml) was added TFA (20 ml). The reaction mixture was stirred at rt for 2 h. Concentrated the product mixture directly, the yellow residue was purified by silica gel chromatography (MeOH/DCM=1-2% as eluent) to give product Raa7 (500 mg, 30%) as light yellow solid.

FKBD Example 10 4-((3-((R)-1-(((S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)morpholine-3-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoic acid (Raa8)

(E)-3-(3,4-dimethoxyphenyl)-1-(3-nitrophenyl)prop-2-en-1-one (3)

To the solution of 3,4-dimethoxybenzaldehyde 1 (60 g, 360 mmol) and 1-(3-nitrophenyl)ethan-1-one 2 (59.6 g, 360 mmol) in MeOH (1100 mL) was added NaOH (15 g) at 0° C. The resulting solution was stirred at rt for 10 h. The precipitate was collected to give compound 3 as a yellow solid (97 g, 86%). [M+Na]⁺=336.1

1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propan-1-one (4)

A solution of 3 (32 g, 110 mmol) and 10% Pd/C (10 g) in THF (120 mL) was hydrogenated with H₂ for 8 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 4 as a white solid (24 g, 76%). [M+H]⁺=286.2

tert-butyl 4-((3-(3-(3,4-dimethoxyphenyl)propanoyl)phenyl)amino)-4-oxobutanoate (5)

To a solution of 4 (12.0 g, 42 mmol) in DCM (30 mL) was added 4-tert-butoxy-4-oxobutanoic acid (8.8 g, 50 mmol), DIPEA (13.6 g, 105 mmol) and HATU (19.2 g, 50 mmol). The mixture was stirred at rt for 16 h. The product was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 5 as a white solid (16 g, 79%). [M+Na]⁺=464.0

tert-butyl (R)-4-((3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenyl)amino)-4-oxobutanoate (6)

A solution of ketone 5 (11.9 g, 26.9 mmol) in dry THF (120 mL) at −20° C. was treated with a solution of (+)-DIPChloride (135 mmol) in heptane (1.7 M, 79 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 5, then quenched with 2,2′-(ethylenedioxy)diethylamine (20 g) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 3:1) to give compound 6 as a light yellow oil (7.9 g, 66%, ee 97%). [M+Na]⁺=466.3

(R)-1-(3-(4-(tert-butoxy)-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)morpholine-3-carboxylate (8)

A solution of 6 (2.36 g, 5.32 mmol) and 7 (2 g, 6.38 mmol) in CH₂Cl₂ (10 mL) was cooled to −20° C. before a solution of DCC (1.65 g, 7.98 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 65 mg, 0.53 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 2:1) to give compound 8 as a light yellow oil (2.5 g, 64%). [M+Na]⁺=761.4

4-((3-((R)-1-(((S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)morpholine-3-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoic acid (Raa8)

A solution of 8 (2.5 g, 3.45 mmol) in CH₂Cl₂ (12 mL) was treated with a solution of 40% TFA in CH₂Cl₂(12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Raa8 (815 mg, 38%) as a pale yellow solid.

FKBD Example 11 4-((3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl) amino)-4-oxobutanoic acid (Raa9)

1-((9H-fluoren-9-yl)methyl) 3-((R)-1-(3-(4-(tert-butoxy)-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl) (S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-1,3-dicarboxylate (3)

A solution of 1 (1.35 g, 3.04 mmol) and 2 (1.95 g, 3.65 mmol) in CH₂Cl₂ (10 mL) was cooled to −20° C. before a solution of DCC (940 mg, 4.56 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 37 mg, 0.3 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 3 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (DCM/MeOH 96:4) to give compound 3 as a white solid (3.0 g, quant.). [M+Na]⁺=981.6

4-((3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-4-oxobutanoic acid (Raa9)

A solution of 3 (1.5 g, 1.56 mmol) in CH₂Cl₂ (12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Raa9 (1.4 g, 99%) as a white solid.

FKBD Example 12 (S)—((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carboxylate (Raa10)

(S)-1-(9H-fluoren-9-yl)methyl 3-((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl) 4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-1,3-dicarboxylate (2)

To the solution of 1 (1.3 g, 1.35 mmol) in DMF (5 mL) was added TBAF (3.2 ml, 1.0 M, 3.18 mmol) at 0° C. The resulting solution was heated to room temperature for 5 h. After this time the reaction mixture was washed with NaHCO₃ (aq., 50 ml*3) and NaCl (aq., 50 ml*3). The organic phase was concentrated. The reaction mixture was purified on silica with DCM/MEOH=50/1 to give 2 (800 mg, 80%) as a colourless oil. [M+H]⁺=738.4

(S)—((R)-1-(3-(4-tert-butoxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carboxylate (Raa10)

A solution of 2 (800 mg, 1.08 mmol) in CHOOH (1.6 mL) was treated with an aqueous solution of formaldehyde (37% in water, 0.8 ml, 1.3 mmol) and allowed to stir at 50° C. for 1 h. After this time the reaction mixture was purified with DCM/MeOH=100/1 give 3 (400 mg, 50%) as a colorless oil. [M+H]⁺=751.9

FKBD Example 13 (S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-3-hydroxy-4-oxobutanoic acid (Raa11)

tert-butyl 3-(3-(3,4-dimethoxyphenyl)propanoyl)phenylcarbamate (2)

To the solution of 1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propan-1-one 1 (8.5 g, 29.79 mmol) in 1,4-dioxane (85 mL) was added (Boc)₂O (9.75 g, 44.68 mmol). The resulting solution was heated to 100° C. for 3 h. The solvent was evaporated and the residue (10.3 g, crude) was used directly for the next step without purification. [M+Na]⁺=408

(R)-tert-butyl 3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylcarbamate (3)

A solution of ketone 2 (10 g, crude) in dry THF (200 mL) at −20° C. was treated with a solution of (+)-DIPChloride in heptane (1.7 M, 76.2 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 2, then quenched with 2,2′-(ethylenedioxy)diethylamine (23.1 g) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 3 as a light yellow oil (8.3 g, 80%). [M+Na]⁺=410

(S)—((R)-1-(3-(tert-butoxycarbonylamino)phenyl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (5)

A solution of 3 (8.3 g, 21.42 mmol) and 4 (8 g, 25.7 mmol) in CH₂Cl₂ (100 mL) was cooled to −20° C. before a solution of DCC (5.3 g, 25.7 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 318 mg, 2.6 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 5 as a light yellow oil (12 g, 83%). [M+Na]⁺=703.3

(S)—((R)-1-(3-aminophenyl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (6)

To a solution of 5 (5 g, 7.34 mmol) in DCM (30 ml) was added TFA (6 ml). The mixture was stirred at 35° C. for 6 h. The solvent was evaporated and the residue (5.0 g, crude) was used directly for the next step without purification. [M+H]⁺=580.8

(S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-3-hydroxy-4-oxobutanoic acid (Raa11)

A solution of 6 (1.0 g, crude) in DCM (20 mL) was added 7 (400 mg, 3.4 mmol) and DMAP (25 mg, 0.2 mmol). The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (DCM/MeOH=10:1) to afford Raa11 (450 mg, 38%) as a white solid.

FKBD Example 14 (S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-2-hydroxy-4-oxobutanoic acid (Raa12)

The synthesis of 6 is the same as Raa11.

(S)—((R)-1-(3-((S)-4-(allyloxy)-3-hydroxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

To a solution of 6 (2 g, 3.44 mmol) in DMF (30 ml) was added 7 (1.2 g, 6.9 mmol) DIPEA (1.33 g, 0.32 mmol) and HATU (1.96 g, 5.16 mmol). The mixture was stirred at rt for 3 h before being diluted with EtOAc. The organic layer was washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by silica-gel column (DCM/MeOH 10:1) to give product 8 as a yellow oil (800 mg, 32%). [M+H]⁺=737.

(S)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-2-hydroxy-4-oxobutanoic acid (Raa12)

A solution of 8 (800 mg, 1.09 mmol) in THF (100 mL) was added N-Methylaniline (232 mg, 2.17 mmol) and Pd(PPh₃)₄(115 mg, 0.1 mmol). The mixture was allowed to react at room temperature under N₂ atmosphere until complete conversion. The reaction mixture was charged to silica-gel flash column directly (DCM/MeOH 10:1) to afford Raa12 (120 mg, 16%) as a white solid.

FKBD Example 15 (S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutanoic acid (Raa13)

(S)-tert-butyl 3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-(3-(3,4-dimethoxyphenyl)propanoyl)phenylamino)-4-oxobutanoate (3)

A solution of 1 (4.0 g, 14.03 mmol), 2 (7.0 g, 16.8 mmol) in DCM (150 mL) was treated with DIPEA (8 ml, 42.1 mmol) and HATU (8.0 g, 21.1 mmol) at 0° C. and allowed to stir at room temperature for 15 h. After this time the reaction mixture was washed with H₂O and extracted with AcOEt (50 ml*3). The organic phase was dried over Na₂SO₄ and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:10) to give compound 3 as a brown oil (9 g, 90%). [M+Na]⁺=700.9

(S)-tert-butyl 3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylamino)-4-oxobutanoate (4)

A solution of ketone 3 (4.7 g, 6.9 mmol) in dry THF (130 mL) at −20° C. was treated with a solution of (+)-DIPChloride (27.7 mmol) in heptane (1.7 M, 16.3 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 3, then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:10) to give compound 4 as a light yellow oil (1.7 g, 40%). [M+Na]⁺=702.8

(S)—((R)-1-(3-((S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-tert-butoxy-4 oxobutanamido) phenyl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (6)

A solution of 4 (1.7 g, 2.5 mmol) and 5 (1.2 g, 3.75 mmol) in CH₂Cl₂ (50 mL) was cooled to −20° C. before a solution of DCC (0.78 g, 3.75 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 30 mg, 0.25 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 6 as a light yellow oil (1.0 g, 50%).

(S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutanoic acid (Raa13)

A solution of 6 (1.0 g, 1.02 mmol) in CH₂Cl₂ (10 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (DCM/MeOH=50/1) to afford Raa13 (401 mg, 42%) as a white solid.

FKBD Example 16 (S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutanoic acid (Raa14)

(S)-tert-butyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-(3-(3,4-dimethoxyphenyl)propanoyl)phenylamino)-4-oxobutanoate (3)

A solution of 1 (4.0 g, 14.03 mmol), 2 (7.0 g, 16.8 mmol) in DCM (150 mL) was treated with DIPEA (8 ml, 42.1 mmol) and HATU (8.0 g, 21.1 mmol) at 0° C. and allowed to stir at room temperature for 15 h. After this time the reaction mixture was washed with H₂O and extracted with AcOEt (50 ml*3). The organic phase was dried over Na₂SO₄ and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:10) to give compound 3 as a brown oil (9 g, 90%). [M+Na]⁺=700.9

(S)-tert-butyl 2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((S)-3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenylamino)-4-oxobutanoate (4)

A solution of ketone 3 (4.0 g, 5.9 mmol) in dry THF (80 mL) at −20° C. was treated with a solution of (+)-DIPChloride (23.6 mmol) in heptane (1.7 M, 14.0 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 3, then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:10) to give compound 4 as a light yellow oil (2.0 g, 50%). [M+Na]⁺=702.8

(S)—((R)-1-(3-((S)-3-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-tert-butoxy-4-oxobutanamido)phenyl)-3-(3,4-dimethoxyphenyl)propyl) 1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (6)

A solution of 4 (2.0 g, 2.9 mmol) and 5 (1.2 g, 3.82 mmol) in CH₂Cl₂ (50 mL) was cooled to −20° C. before a solution of DCC (0.91 g, 4.11 mmol) in CH₂Cl₂(5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 35 mg, 0.29 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 6 as a light yellow oil (1.0 g, 50%).

(S)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-4-(3-((R)-1-((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyloxy)-3-(3,4-dimethoxyphenyl)propyl)phenylamino)-4-oxobutanoic acid (Raa14)

A solution of 6 (1.0 g, 1.02 mmol) in CH₂Cl₂ (10 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (DCM/MeOH=50/1) to afford Raa14 (367 mg, 42%) as a white solid.

FKBD Example 17 (2S,3S)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2,3-dihydroxy-4-oxobutanoic acid (Raa15)

(3R, 4S)-3, 4-dihydroxydihydrofuran-2, 5-dione (2)

To the solution of (2R,3S)-2,3-dihydroxysuccinic acid 1 (10 g, 66.6 mmol) in DCM (100 mL) was added 2,2,2-trifluoroacetic anhydride (27.9 g, 133.2 mmol) at 25° C. The resulting solution was stirred at room temperature for 12 h. The mixture was concentrated in vacuum. The crude product was washed with petroleum ether (100 mL) to afford 2 (6 g, 68%) as a white solid.

(2S,3S)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2,3-dihydroxy-4-oxobutanoic acid (Raa15)

A mixture of 6 (2 g, 3.4 mmol), 2 (0.896 g, 6.8 mmol) and DMAP (80 mg, 0.68 mmol) in THF (60 mL) were stirred at 50° C. for 6 h. The mixture was filtered and concentrated in vacuum. The resulting residue was purified by prep-HPLC to afford Raa15 (476 mg, 19%) as a white solid.

FKBD Example 18 3-((((9H-fluoren-9-yl)methoxy)amino)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2-hydroxy-4-oxobutanoic acid (Raa16)

2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-hydroxysuccinic acid (2)

To a solution of 1 (10 g, 67.1 mmol) in 1,4-dioxane (150 ml) was added 10% NaCO₃(aq) 250 ml, and then FmocCl in 1,4-dioxane (150 ml) was dropwisely added at 0° C. The reaction mixture was stirred at 0° C. for 10 min, and then raised to rt and stirred for another 4 h. The product mixture was poured into water (500 ml), extracted with EA (200 ml) 3 times. Adjusted the hydrous layer to pH=2-3 by 2M HCl, and then extracted with DCM (200 ml) 3 times, combined the organic layer, washed with brine (200 ml) 3 times, dried over Na₂SO₄, filtered and concentrated to get product 2 (22 g, 88%) as white solid. [M+Na]⁺=394

2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetic acid (4)

To a solution of 2 (5 g, 13.5 mmol) in EA (50 ml) was added 3 (14 g, 135 mmol) and PTSA (0.46 g 2.7 mmol). The reaction mixture was refluxed for 16 h. The product mixture was concentrated directly, and the brown residue was purified by silica gel chromatography (EA/PE=10-50% as eluent) to give 4 (3.8 g, 68.6%) as white solid. [M+Na]⁺=434

(1R)-1-(3-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetamido)phenyl)-3-(3,4-dimethoxyphenyl(2S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (6)

To a solution of 4 (2.2 g, 5.4 mmol) in DMF (150 ml) was added HATU (3 g, 8 mmol) and DIEA (1.38 g, 10.8 mmol). 5 (2.6 g 4.5 mmol) was added at last. The reaction mixture was stirred at rt for 1 h. Poured the product mixture into water (300 ml), extracted with DCM (100 ml*3), combined the organic phase and washed with brine (100 ml*5). Dried over Na₂SO₄, filtered and concentrated to get the crude. Purified by silica gel chromatography (Methanol/DCM=0-2% as eluent) to give compound 6 (3.7 g, 71%) as white solid. [M+Na]⁺=996

3-((((9H-fluoren-9-yl)methoxy)amino)-4-((3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenyl)amino)-2-hydroxy-4-oxobutanoic acid (Raa16)

To a solution of 6 (3.7 g, 38 mmol) in THF/H₂O (10 ml/10 ml) was added THF (40 ml). The reaction mixture was stirred at rt for 1 h. The product mixture was evaporated directly, and the residue was purified by silica gel chromatography (HCOOH/DCM=0-5% as eluent) to give compound Raa16 (500 mg, 14%) as light yellow solid.

FKBD Example 19 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae1)

(E)-1-(3-hydroxyphenyl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (3)

To the solution of 3,4,5-trimethoxybenzaldehyde 1 (5 g, 25.5 mmol) and 3′-hydroxyacetophenone 2 (3.47 g, 25.5 mmol) in EtOH (50 mL) was added a solution of 10% aqueous NaOH (41 mL, 4.1 g, 101.9 mmol) at 0° C. The resulting solution was heated to 65° C. for 2 h. The solvent was evaporated and the residue (5.5 g, crude) was used directly for the next step without purification. [M+H]⁺=314.9.

3-(1-hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenol (4)

A solution of 3 (5.5 g, crude) and 10% Pd/C (2 g) in THF (40 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated to a solid (5.96 g, crude). [M+H—H₂O]⁺=300.9

tert-butyl 2-(3-(1-hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)acetate (5)

A solution of 4 (5.96 g, 18.8 mmol, crude) and K₂CO₃ (3.12 g, 22.6 mmol) in DMF (30 mL) was treated with tert-butyl bromoacetate (3.68 g, 18.8 mmol) and allowed to stir at room temperature for 5 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated. The crude product was purified by prep-HPLC to give 5 (4.65 g, 42% (3 steps)) as a yellow solid. [M+Na]⁺=454.8

tert-butyl 2-(3-(3-(3,4,5-trimethoxyphenyl)propanoyl)phenoxy)acetate (6)

A solution of 5 (4.65 g, 10.75 mmol) in CH₂Cl₂(110 mL) was treated with Dess-Martin periodinane (11.4 g, 26.88 mmol) and allowed to stir at room temperature for 3 h before being quenched with a solution of 10% aqueous NaS₂O₃. The solution was extracted with CH₂Cl₂ twice. The combined organic layers were washed by sat. NaHCO₃, brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a white solid (4.5 g, 97%). [M+Na]⁺=453.2

tert-butyl (R)-2-(3-(1-hydroxy-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)acetate (7)

A solution of ketone 6 (3.98 g, 9.25 mmol) in dry THF (40 mL) at −20° C. was treated with a solution of (+)-DIPChloride (18.5 mmol) in heptane (1.7 M, 10.88 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 7 as a light yellow oil (2.8 g, 70%, ee>99%). [M+Na]⁺=455.2

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (1.85 g, 4.3 mmol) and 8 (2 g, 6.4 mmol) in CH₂Cl₂(15 mL) was cooled to −20° C. before a solution of DCC (1.3 g, 6.4 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 52.3 mg, 0.43 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 9 as a light yellow oil (2.5 g, 80%). [M+Na]⁺=748.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae1)

A solution of 9 (2.5 g, 3.44 mmol) in CH₂Cl₂ (11.5 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (11.5 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae1 (969 mg, 42%) as a white solid.

FKBD Example 20 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae2)

(E)-1-(3-hydroxyphenyl)-3-(2,3,4-trimethoxyphenyl)prop-2-en-1-one (3)

To the solution of 2,3,4-trimethoxybenzaldehyde 1 (5 g, 25.5 mmol) and 3′-hydroxyacetophenone 2 (3.47 g, 25.5 mmol) in EtOH (30 mL) was added a solution of 10% aqueous NaOH (41 mL, 4.1 g, 101.9 mmol) at 0° C. The resulting solution was heated to 65° C. for 3 h. The solvent was evaporated and the residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 3 as a yellow oil (6.6 g, 83%). [M+H]⁺=314.9

3-(1-hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenol (4)

A solution of 3 (6.6 g, 21 mmol) and 10% Pd/C (3 g) in THF (30 mL) was hydrogenated with H₂ for 16 h at room temperature. The reaction mixture was then filtered and concentrated to a colorless oil (8 g, crude). [M+H—H₂O]⁺=301.0

tert-butyl 2-(3-(1-hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)acetate (5)

A solution of 4 (8 g, 25 mmol, crude) and K₂CO₃ (4.19 g, 30 mmol) in DMF (30 mL) was treated with tert-butyl bromoacetate (5.92 g, 30 mmol) and allowed to stir at room temperature for 6 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a colorless oil (8.2 g, 90% (2 steps)). [M+H—H₂O-tBu]⁺=358.8

tert-butyl 2-(3-(3-(2,3,4-trimethoxyphenyl)propanoyl)phenoxy)acetate (6)

A solution of 5 (5.75 g, 13.29 mmol) in CH₂Cl₂ (30 mL) was treated with Dess-Martin periodinane (11.28 g, 26.59 mmol) and allowed to stir at room temperature for 2 h before being quenched with a solution of 10% aqueous NaS₂O₃. The solution was extracted with CH₂Cl₂ twice. The combined organic layers were washed by sat. NaHCO₃, brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a yellow oil (5 g, 87%).

tert-butyl (R)-2-(3-(1-hydroxy-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)acetate (7)

A solution of ketone 6 (5 g, 11.61 mmol) in dry THF (50 mL) at −20° C. was treated with a solution of (+)-DIPChloride (23.23 mmol) in heptane (1.7 M, 13.66 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (3.4 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 7 as a light yellow oil (4 g, 80%, ee 83%). [M+H—H₂O-tBu]⁺=358.9

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4,5-trimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (2 g, 4.62 mmol) and 8 (2.16 g, 6.93 mmol) in CH₂Cl₂ (23 mL) was cooled to −20° C. before a solution of DCC (1.43 g, 6.93 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 57 mg, 0.46 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 9 as a light yellow oil (2.13 g, 64%). [M+Na]⁺=748.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,4-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae2)

A solution of 9 (2.13 g, 2.93 mmol) in CH₂Cl₂(12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae2 (508 mg, 25%) as a pale yellow solid.

FKBD Example 21 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae3)

(E)-1-(3-hydroxyphenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (3)

To the solution of 2,4,5-trimethoxybenzaldehyde 1 (4.5 g, 22.96 mmol) and 3′-hydroxyacetophenone 2 (3.1 g, 22.96 mmol) in EtOH (50 mL) was added a solution of 10% aqueous KOH (15 mL, 5.1 g, 91.84 mmol) at 0° C. The resulting solution was heated to 60° C. for 4 h. The solvent was evaporated and the residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 3 as a yellow oil (5.9 g, 82%). [M+H]⁺⁼315.1

tert-butyl (E)-2-(3-(3-(2,4,5-trimethoxyphenyl)acryloyl)phenoxy)acetate (4)

A solution of 3 (7.4 g, 23.6 mmol) and K₂CO₃ (3.9 g, 28.3 mmol) in DMF (200 mL) was treated with tert-butyl bromoacetate (5.5 g, 28.3 mmol) and allowed to stir at room temperature for 4 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 4 as a colorless oil (8 g, 80%). [M+H]⁺=429.3

tert-butyl 2-(3-(3-(2,4,5-trimethoxyphenyl)propanoyl)phenoxy)acetate (5)

A solution of 4 (8 g, 18.69 mmol) and 10% Pd/C (1 g) in THF (200 mL) was hydrogenated with H₂ for 8 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 5 as a colorless oil (6 g, 75%). [M+Na]⁺=453.2

tert-butyl (R)-2-(3-(1-hydroxy-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)acetate (6)

A solution of ketone 5 (6 g, 13.95 mmol) in dry THF (60 mL) at −20° C. was treated with a solution of (+)-DIPChloride (41.86 mmol) in heptane (1.7 M, 24.6 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 5, then quenched with 2,2′-(ethylenedioxy)diethylamine (5.9 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 6 as a light yellow oil (5.5 g, 92%, ee>99%).). [M+Na]⁺=455.2

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,4,5-trimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

A solution of 6 (1.85 g, 4.28 mmol) and 7 (2 g, 6.42 mmol) in CH₂Cl₂ (10 mL) was cooled to −20° C. before a solution of DCC (1.33 g, 6.42 mmol) in CH₂Cl₂(5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 52 mg, 0.43 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 8 as a light yellow oil (2.35 g, 76%). [M+Na]⁺=747.9

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,4,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae3)

A solution of 8 (2.35 g, 3.24 mmol) in CH₂Cl₂ (12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae3 (815 mg, 37%) as a pale yellow solid.

FKBD Example 22 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae4)

(E)-1-(3-hydroxyphenyl)-3-(2,3,5-trimethoxyphenyl)prop-2-en-1-one (3)

To the solution of 2,3,5-trimethoxybenzaldehyde 1 (6 g, 30.6 mmol) and 1-(3-hydroxyphenyl)ethan-1-one 2 (4.2 g, 30.6 mmol) in EtOH (50 mL) was added a solution of 10% aqueous NaOH (50 mL, 122.4 mmol) at 0° C. The resulting solution was stirred at room temperature for 12 h. The solution was adjusted to pH 4 by added 4M aqueous HCl dropwise at 0° C., generated a large of yellow solid. Then the mixture was filtered and the solid was washed with water (50 mL) to afford 3 (5.5 g, 57%) as a yellow solid. [M+H]⁺=315.2.

tert-butyl (E)-2-(3-(3-(2,3,5-trimethoxyphenyl)acryloyl)phenoxy) acetate (4)

A solution of 3 (5.5 g, 17.4 mmol) and K₂CO₃ (4.82 g, 34.9 mmol) in DMF (40 mL) was treated with tert-butyl bromoacetate (4.06 g, 20.9 mmol) and allowed to stir at room temperature for 12 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated. The mixture was filtered and the solid was washed with water (30 mL). The crude product was washed with petroleum ether (50 mL) to give 4 (7 g, 93%) as a yellow solid. [M+H]⁺=428.8

tert-butyl 2-(3-(3-(2,3,5-trimethoxyphenyl)propanoyl)phenoxy)acetate (5)

A solution of 4 (7 g, 11.68 mmol) and 10% Pd/C (1 g) in THF (100 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was purified by column chromatography on silica gel to give 5 (3.5 g, 50%) as a yellow oil. [M+Na]⁺=452.9.

tert-butyl (R)-2-(3-(1-hydroxy-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)acetate (6)

A solution of ketone 5 (3.5 g, 8.14 mmol) in dry THF (30 mL) at −20° C. was treated with a solution of (+)-DIPChloride (16.2 mmol) in heptane (1.7 M, 9.5 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (2.4 g) by forming an insoluble complex. After stirring at room temperature for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:4) to give compound 6 (2.2 g, 63%, ee 97% vs racemate) as a light yellow oil. [M+Na]⁺=454.9

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,3,5-trimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

A solution of 6 (2.2 g, 5.09 mmol) and 8 (1.89 g, 6.1 mmol) in CH₂Cl₂ (15 mL) was cooled to −20° C. before a solution of DCC (1.36 g, 6.6 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (62 mg, 0.5 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 8 (1.8 g, 49%) as a light yellow oil. [M+Na]⁺=748.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,5-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae4)

A solution of 8 (1.8 g, 2.48 mmol) in CH₂Cl₂(10 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae4 (652 mg, 39%) as a faint yellow solid.

FKBD Example 23 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae5)

(E)-1-(3-hydroxyphenyl)-3-(2,3,6-trimethoxyphenyl)prop-2-en-1-one (3)

To the solution of 2,3,6-trimethoxybenzaldehyde 1 (5 g, 25.48 mmol) and 3′-hydroxyacetophenone 2 (3.47 g, 25.48 mmol) in EtOH (40 mL) was added a solution of 40% aqueous KOH (15 mL, 5.7 g, 101.92 mmol) at 0° C. The resulting solution was reacted at room temperature for 4 h. The solvent was evaporated and the residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 3 as a yellow oil (4 g, 50%). [M+H]⁺=315.2

tert-butyl (E)-2-(3-(3-(2,3,6-trimethoxyphenyl)acryloyl)phenoxy)acetate (4)

A solution of 3 (3.5 g, 11.15 mmol) and K₂CO₃ (1.85 g, 13.37 mmol) in DMF (60 mL) was treated with tert-butyl bromoacetate (2.6 g, 13.37 mmol) and allowed to stir at room temperature for 4 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:4) to give compound 4 as a yellow oil (4.7 g, 98%). [M+H]⁺⁼429.0

tert-butyl 2-(3-(3-(2,3,6-trimethoxyphenyl)propanoyl)phenoxy)acetate (5)

A solution of 4 (4.6 g, 10.75 mmol) and 10% Pd/C (0.5 g) in THF (70 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 5 as a colorless oil (2.9 g, 63%). [M+Na]⁺=453.3

tert-butyl (R)-2-(3-(1-hydroxy-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)acetate (6)

A solution of ketone 5 (2.9 g, 6.7 mmol) in dry THF (30 mL) at −20° C. was treated with a solution of (+)-DIPChloride (13.48 mmol) in heptane (1.7 M, 7.9 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 5, then quenched with 2,2′-(ethylenedioxy)diethylamine (1.96 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 6 as a light yellow oil (2.4 g, 83%, ee>99%). [M+Na]⁺=454.9

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2,3,6-trimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine 2-carboxylate (8)

A solution of 6 (1.46 g, 3.45 mmol) and 7 (1.6 g, 5.17 mmol) in CH₂Cl₂ (18 mL) was cooled to −20° C. before a solution of DCC (1.065 g, 5.17 mmol) in CH₂Cl₂(5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 43 mg, 0.35 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 8 as a light yellow oil (1.7 g, 68%).

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2,3,6-trimethoxyphenyl)propyl)phenoxy)acetic acid (Rae5)

A solution of 8 (1.7 g, 2.34 mmol) in CH₂Cl₂ (12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae5 (494 mg, 31%) as a pale yellow solid.

FKBD Example 24 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae9)

2-(tert-butoxy)-3,4-dimethoxybenzaldehyde (2)

To a solution of 2-hydroxy-3,4-dimethoxybenzaldehyde 1 (2.77 g, 15.2 mmol) in anhydrous toluene (30 mL) was added 1,1-di-tert-butoxy-N,N-dimethylmethanamine 2 (29.1 mL, 122 mmol) under Ar. atmosphere. The mixture was stirred at 80° C. for 6 h, then the solvent was evaporated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 2 as a yellow solid (2.965 g, 82%). [M+Na]⁺=261.1

(E)-3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (4)

To the solution of 2 (2.965 g, 12.4 mmol) and 3′-hydroxyacetophenone 3 (2.03 g, 14.9 mmol) in EtOH (50 mL) was added a solution of 40% aqueous KOH (6.98 g, 49.8 mmol) at 0° C. The resulting solution was stirred at 60° C. for 4 h. The solution was poured into water and acidified to pH 4 with a 1 M HCl aqueous solution, extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 4 as a yellow oil (3.2 g, 72%). [M+Na]⁺=378.9

tert-butyl (E)-2-(3-(3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)acryloyl)phenoxy)acetate (5)

A solution of 4 (3.2 g, 9 mmol) and K₂CO₃ (1.49 g, 10.8 mmol) in DMF (30 mL) was treated with tert-butyl bromoacetate (1.58 mL, 10.8 mmol) and allowed to stir at room temperature for 5 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:4) to give compound 5 as a yellow oil (4 g, 95%). [M+Na]⁺=493.3

tert-butyl 2-(3-(3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate (6)

A solution of 5 (4 g, 8.5 mmol) and 10% Pd/C (0.8 g) in THF (50 mL) was hydrogenated with H₂ for 3 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a colorless oil (2.878 g, 72%). [M+Na]⁺=495.3

tert-butyl (R)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (7)

A solution of ketone 6 (2.878 g, 6.1 mmol) in dry THF (30 mL) at −20° C. was treated with a solution of (+)-DIPChloride (24.4 mmol) in heptane (1.7 M, 14.3 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (3.6 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 7 as a light yellow oil (2 g, 70%, ee>99%). [M+Na]⁺=497.0

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (1.8 g, 3.793 mmol) and 8 (1.77 g, 5.69 mmol) in CH₂Cl₂ (13 mL) was cooled to −20° C. before a solution of DCC (1.17 g, 5.69 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 46 mg, 0.379 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 9 as a light yellow oil (2.5 g, 86%). [M+Na]⁺=790.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae9)

A solution of 9 (2.5 g, 3.26 mmol) in CH₂Cl₂(12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae9 (636 mg, 30%) as a pale yellow solid.

FKBD Example 25 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae10)

3-(tert-butoxy)-4,5-dimethoxybenzaldehyde (2)

To a solution of 3-hydroxy-4,5-dimethoxybenzaldehyde 1 (2.77 g, 15.2 mmol) in anhydrous toluene (30 mL) was added 1,1-di-tert-butoxy-N,N-dimethylmethanamine 2 (29.1 mL, 122 mmol) under Ar. atmosphere. The mixture was stirred at 80° C. for 6 h, then the solvent was evaporated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 2 as a yellow solid (3.126 g, 86%). [M+H]⁺=239.0

(E)-3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (4)

To the solution of 2 (3.126 g, 13 mmol) and 3′-hydroxyacetophenone 3 (2.14 g, 15.7 mmol) in EtOH (30 mL) was added a solution of 40% aqueous KOH (7.36 g, 52 mmol) at 0° C. The resulting solution was stirred at 60° C. for 4 h. The solution was poured into water and acidified to pH 4 with a 1 M HCl aqueous solution, extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 4 as a yellow oil (2.489 g, 54%). [M+H]⁺=357.0

tert-butyl (E)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)acryloyl)phenoxy)acetate (5)

A solution of 4 (2.489 g, 6.98 mmol) and K₂CO₃ (1.16 g, 8.38 mmol) in DMF (30 mL) was treated with tert-butyl bromoacetate (1.2 mL, 8.38 mmol) and allowed to stir at room temperature for 5 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:4) to give compound 5 as a yellow oil (3.1 g, 95%). [M+H]⁺=471.0

tert-butyl 2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)propanoyl)phenoxy)acetate (6)

A solution of 5 (3.1 g, 6.59 mmol) and 10% Pd/C (0.5 g) in THF (50 mL) was hydrogenated with H12 for 3 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a colorless oil (2.88 g, 93%). [M+Na]⁺=495.3

tert-butyl (R)-2-(3-(3-(3-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (7)

A solution of ketone 6 (2.868 g, 6.07 mmol) in dry THF (30 mL) at −20° C. was treated with a solution of (+)-DIPChloride (12.1 mmol) in heptane (1.7 M, 7.1 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (3.6 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 7 as a light yellow oil (2.03 g, 70%, ee>99% vs racemate). [M+Na]⁺=497.3

(R)-1-(3-(3-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (2.03 g, 4.3 mmol) and 8 (2 g, 6.4 mmol) in CH₂Cl₂ (43 mL) was cooled to −20° C. before a solution of DCC (1.3 g, 6.4 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 52.3 mg, 0.43 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 9 as a light yellow oil (2.5 g, 76%). [M+Na]⁺=790.3

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae10)

A solution of 9 (2.5 g, 3.26 mmol) in CH₂Cl₂ (12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae100 (1.334 g, 62%) as a pale yellow solid.

FKBD Example 26 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae11)

2-(tert-butoxy)-4,5-dimethoxybenzaldehyde (2)

To a solution of 2-hydroxy-4,5-dimethoxybenzaldehyde 1 (3 g, 16.5 mmol) in anhydrous toluene (15 mL) was added 1,1-di-tert-butoxy-N,N-dimethylmethanamine 2 (31.6 mL, 132 mmol) under Ar. atmosphere. The mixture was stirred at 80° C. for 6 h, then the solvent was evaporated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 2 as a yellow solid (3.875 g, 99%). [M+Na]⁺=261.2

(E)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (4)

To the solution of 2 (3.875 g, 16.3 mmol) and 3′-hydroxyacetophenone 3 (2.436 g, 17.9 mmol) in EtOH (50 mL) was added a solution of 40% aqueous KOH (8.5 mL, 3.65 g, 65.2 mmol) at 0° C. The resulting solution was stirred at 60° C. for 4 h. The solution was poured into water and acidified to pH 4 with a 1M HCl aqueous solution, extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 4 as a yellow oil (5.2 g, 90%). [M+H]⁺⁼357.2

tert-butyl (E)-2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)acryloyl)phenoxy)acetate (5)

A solution of 4 (5.2 g, 14.59 mmol) and K₂CO₃ (2.4 g, 17.5 mmol) in DMF (50 mL) was treated with tert-butyl bromoacetate (2.55 mL, 17.5 mmol) and allowed to stir at room temperature for 5 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were washed by brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:4) to give compound 5 as a yellow oil (6 g, 88%). [M+H]⁺=471.0

tert-butyl 2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propanoyl)phenoxy)acetate (6)

A solution of 5 (6 g, 12.75 mmol) and 10% Pd/C (1 g) in THF (70 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a colorless oil (4.5 g, 75%). [M+Na]⁺=495.3

tert-butyl (R)-2-(3-(3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (7)

A solution of ketone 6 (4.5 g, 9.5 mmol) in dry THF (45 mL) at −20° C. was treated with a solution of (+)-DIPChloride (19 mmol) in heptane (1.7 M, 11.2 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 7 as a light yellow oil (3.2 g, 70%, ee>99% vs racemate). [M+Na]⁺=496.7

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-(tert-butoxy)-4,5-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (1.93 g, 4.07 mmol) and 8 (1.9 g, 6.103 mmol) in CH₂Cl₂ (43 mL) was cooled to −20° C. before a solution of DCC (1.26 g, 6.103 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 50 mg, 0.407 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 9 as a light yellow oil (2.1 g, 67%). [M+Na]⁺=790.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-hydroxy-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae11)

A solution of 9 (2.1 g, 2.73 mmol) in CH₂Cl₂(12 mL) was treated with a solution of 40% TFA in CH₂Cl₂(12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae11 (638 mg, 23%) as a pale yellow solid.

FKBD Example 27 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae12)

(E)-3-(3-fluoro-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3)

To the solution of 3-fluoro-4,5-dimethoxybenzaldehyde 1 (4.5 g, 24.4 mmol) and 1-(3-hydroxyphenyl)ethan-1-one 2 (3.3 g, 24.4 mmol) in EtOH (60 mL) was added a solution of 10% aqueous NaOH (40 mL, 97.6 mmol) at 0° C. The resulting solution was stirred at room temperature for 12 h. The solution was adjusted to pH 4 by added 4M aqueous HCl dropwise at 0° C., generated a large of yellow solid. Then the mixture was filtered and the solid was washed with water (50 mL) to afford 3 (4 g, 54%) as a yellow solid. [M+H]⁺=303.1

tert-butyl (E)-2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)acryloyl)phenoxy)acetate (4)

A solution of 3 (4 g, 13.2 mmol) and K₂CO₃ (3.65 g, 26.4 mmol) in DMF (30 mL) was treated with tert-butyl bromoacetate (3.08 g, 15.8 mmol) and allowed to stir at room temperature for 5 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated. The mixture was filtered and the solid was washed with water (30 mL). The crude product was purified by column chromatography on silica gel (AcOEt/PE 1:4) to give 4 (5.2 g, 94%) as a yellow solid. [M+Na]⁺=438.7

tert-butyl 2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (5)

A solution of 4 (5.2 g, 12.5 mmol) and 10% Pd/C (1 g) in THF (100 mL) was hydrogenated with H₂ for 2 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was purified by column chromatography on silica gel to give 5 (5 g, 96%) as a yellow oil. [M+Na]⁺=443.2

tert-butyl 2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)propanoyl)phenoxy)acetate (6)

A solution of 5 (5 g, 11.9 mmol) in CH₂Cl₂ (100 mL) was treated with Dess-Martin periodinane (15.2 g, 36 mmol) and allowed to stir at room temperature for 2 h before being quenched with a solution of 10% aqueous NaS₂O₃. The solution was extracted with CH₂Cl₂ twice. The combined organic layers were washed by sat. NaHCO₃, brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a white solid (4 g, 80%). [M+Na]⁺=441.2

tert-butyl (R)-2-(3-(3-(3-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (7)

A solution of ketone 6 (4 g, 9.56 mmol) in dry THF (30 mL) at −20° C. was treated with a solution of (+)-DIPChloride (19.1 mmol) in heptane (1.7 M, 11.2 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (2.8 g) by forming an insoluble complex. After stirring at room temperature for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:4) to give compound 6 (2.2 g, 55%, ee>99%) as a light yellow oil. [M+Na]⁺=442.7

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (2.2 g, 5.23 mmol) and 8 (1.80 g, 5.76 mmol) in CH₂Cl₂ (20 mL) was cooled to −20° C. before a solution of DCC (1.4 g, 6.79 mmol) in CH₂Cl₂ (10 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (63 mg, 0.52 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 9 (1.8 g, 48%) as a light yellow oil. [M+Na]⁺=736.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae12)

A solution of 9 (1.8 g, 2.52 mmol) in CH₂Cl₂(10 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae12 (590 mg, 35%) as a faint yellow solid.

FKBD Example 28 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae13)

(E)-3-(2-fluoro-4,5-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3)

To the solution of 2-fluoro-4,5-dimethoxybenzaldehyde 1 (4.5 g, 24.4 mmol) and 1-(3-hydroxyphenyl)ethan-1-one 2 (3.3 g, 24.4 mmol) in EtOH (60 mL) was added a solution of 10% aqueous NaOH (40 mL, 97.6 mmol) at 0° C. The resulting solution was stirred at 65° C. for 6 h. The solution was adjusted to pH 4 by added 4M aqueous HCl dropwise at 0° C., generated a large of yellow solid. Then the mixture was filtered and the solid was washed with water (50 mL) to afford 3 (7 g, 94%) as a yellow solid. [M+H]⁺=302.8

3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenol (4)

A solution of 3 (7 g, 23.1 mmol) and 10% Pd/C (2 g) in THF (150 mL) was hydrogenated with H₂ for 12 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was purified by column chromatography on silica gel to give 4 (7 g, 98%) as a yellow oil. [M+Na]⁺=328.8

tert-butyl 2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (5)

A solution of 4 (7 g, 23.1 mmol) and K₂CO₃ (7 g, 50.6 mmol) in DMF (200 mL) was treated with tert-butyl bromoacetate (6.7 g, 34.5 mmol) and allowed to stir at room temperature for 24 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated. The mixture was filtered and the solid was washed with water (300 mL). The crude product was purified by column chromatography on silica gel (AcOEt/PE 1:6) to give 5 (8 g, 82%) as a yellow solid. [M+Na]⁺=443.2

tert-butyl 2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)propanoyl)phenoxy)acetate (6)

A solution of 5 (8 g, 19 mmol) in CH₂Cl₂ (100 mL) was treated with Dess-Martin periodinane (16 g, 38 mmol) and allowed to stir at room temperature for 2 h before being quenched with a solution of 10% aqueous NaS₂O₃. The solution was extracted with CH₂Cl₂ twice. The combined organic layers were washed by sat. NaHCO₃, brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a yellow solid (6.3 g, 78%). [M+Na]⁺=440.7

tert-butyl (R)-2-(3-(3-(2-fluoro-4,5-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (7)

A solution of ketone 6 (6.3 g, 15.07 mmol) in dry THF (60 mL) at −20° C. was treated with a solution of (+)-DIPChloride (45.2 mmol) in heptane (1.7 M, 26.5 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (6.6 g) by forming an insoluble complex. After stirring at room temperature for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 7 (4.3 g, 68%, ee>99%) as a light yellow oil. [M+Na]⁺=443.2

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (9)

A solution of 7 (1.6 g, 3.81 mmol) and 8 (1.77 g, 5.71 mmol) in CH₂Cl₂ (20 mL) was cooled to −20° C. before a solution of DCC (1.17 g, 5.71 mmol) in CH₂Cl₂ (10 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (50 mg, 0.38 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 9 (1.7 g, 62%) as a light yellow oil. [M+Na]⁺=736.4

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-4,5-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae13)

A solution of 9 (1.7 g, 2.38 mmol) in CH₂Cl₂ (10 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae13 (520 mg, 33%) as a faint yellow solid.

FKBD Example 29 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae14)

(E)-3-(2-fluoro-3,4-dimethoxyphenyl)-1-(3-hydroxyphenyl)prop-2-en-1-one (3)

To the solution of 1 (5.0 g, 27.17 mmol) and 2 (4.10 g, 29.89 mmol) in EtOH (150 mL) was added a solution of 40% aqueous KOH (15.22 g, 108.70 mmol) at 0° C. The resulting solution was heated to 35° C. for 2 h. The solvent was evaporated and the residue (4.8 g 58%) was used directly for the next step without purification. [M+H]⁺=303.0

(E)-tert-butyl 2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)acryloyl)phenoxy)acetate (4)

A solution of 3 (5.0 g, 16.55 mmol, crude) and K₂CO₃ (2.74 g, 19.87 mmol) in DMF (40 mL) was treated with tert-butyl bromoacetate (3.9 g, 19.87 mmol) and allowed to stir at room temperature for 5 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated. The mixture was filtered and the solid was washed with water (30 mL). The crude product was purified by column chromatography on silica gel to give 4 (6.0 g, 80%) as a yellow solid. [M+Na]⁺=439.2

tert-butyl 2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate (5)

A solution of 4 (4.0 g, 9.62 mmol) and 10% Pd/C (1.0 g) in THF (150 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was purified by column chromatography on silica gel to give 5 (2.8 g, 70%) as a yellow oil. [M+Na]⁺=440.8

tert-butyl (R)-2-(3-(3-(2-fluoro-3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (6)

A solution of ketone 5 (2.8 g, 6.7 mmol) in dry THF (30 mL) at −20° C. was treated with a solution of (+)-DIPChloride (26.8 mmol) in heptane (1.7 M, 15.7 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (3.96 g) by forming an insoluble complex. After stirring at room temperature for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:4) to give compound 6 (1.3 g, 46%, ee>99%) as a light yellow oil. [M+Na]⁺=442.7

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

A solution of 6 (1.3 g, 3.09 mmol) and 7 (1.25 g, 4.02 mmol) in CH₂Cl₂ (15 mL) was cooled to −20° C. before a solution of DCC (0.83 g, 4.02 mmol) in CH₂Cl₂(5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (40 mg, 0.31 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 8 (1.4 g, 63%) as a light yellow oil. [M+Na]⁺=736.3

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(2-fluoro-3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae14)

A solution of 8 (1.4 g, 1.96 mmol) in CH₂Cl₂ (10 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae14 (585 mg, 45%) as a faint yellow solid.

FKBD Example 30 2-(3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae16)

(E)-3-(3,4-dimethoxyphenyl)-1-(3-hydroxyphenylprop-2-en-1-one (3)

To the solution of 3,4-dimethoxybenzaldehyde 1 (17.6 g, 105.8 mmol) and 3′-hydroxyacetophenone 2 (12 g, 88.2 mmol) in EtOH (160 mL) was added a solution of 40% aqueous KOH (44 mL, 20 g, 352.8 mmol) at 0° C. The resulting solution was stirred at rt for 2 h, before being poured into ice-H₂O, the solution was acidified with 1M HCl solution and extracted with EtOAc. The combined organic layers were dried over Na₂SO₄ and concentrated in vacuo. The residue was recrystallized from EtOAc-PE to give the pale yellow powder (23 g, 92%). [M+H]⁺=285.2

3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenol (4)

A solution of 3 (16 g, 56.3 mmol) and 10% Pd/C (1.6 g) in THF (150 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated to a solid (16.3 g, quant.). [M+Na]⁺=311.2

tert-butyl 2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (5)

A solution of 4 (16.3 g, 56.53 mmol) and K₂CO₃ (9.4 g, 67.83 mmol) in DMF (150 mL) was treated with tert-butyl bromoacetate (9.9 mL, 67.83 mmol) and allowed to stir at room temperature for 5 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated (20 g, 88%). [M+Na]⁺=424.9.

tert-butyl 2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate (6)

A solution of 5 (20 g, 49.7 mmol) in CH₂Cl₂(400 mL) was treated with Dess-Martin periodinane (63 g, 149 mmol) and allowed to stir at room temperature for 3 h before being quenched with a solution of 10% aqueous NaS₂O₃. The solution was extracted with CH₂Cl₂ twice. The combined organic layers were washed by sat. NaHCO₃, brine, dried over Na₂SO₄ and concentrated in vacuo. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 as a white solid (11 g, 55%). [M+Na]⁺=423.3.

tert-butyl (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (7)

A solution of ketone 6 (11.156 g, 27.9 mmol) in dry THF (100 mL) at −20° C. was treated with a solution of (+)-DIPChloride (83.6 mmol) in heptane (1.7 M, 49 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (11.5 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 7 as a light yellow oil (6.3 g, 58%, ee>99%). [M+Na]⁺=425.3.

1-((9H-fluoren-9-yl)methyl) 3-((R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl) (S)-4-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-1,3-dicarboxylate (9)

A solution of 7 (1.224 g, 3 mmol) and 8 (2.44 g, 4.56 mmol) in CH₂Cl₂ (10 mL) was cooled to −20° C. before a solution of DCC (0.94 g, 4.56 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 37 mg, 0.3 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 9 as a light yellow oil (1.8 g, 70%). [M+Na]⁺=940.7.

2-(3-((R)-1-(((S)-4-(((9H-fluoren-9-yl)methoxy)carbonyl)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae16)

A solution of 9 (1.8 g, 1.96 mmol) in CH₂Cl₂ (11.5 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (11.5 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae16 (964 mg, 57%) as a white solid.

FKBD Example 31 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae17)

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperazine-2-carboxylate (2)

To the solution of 1 (1.0 g, 1.09 mmol) in DMF (5 mL) was added TBAF (2.5 ml, 1.0 M, 2.55 mmol) at 0° C. The resulting solution was warmed to room temperature for 5 h. After this time the reaction mixture was diluted with DCM and washed with sat. NaHCO₃ aqueous solution and brine. The organic layer was concentrated in vacuo, the residue was purified by silica-gel flash column chromatography (DCM/MeOH 50:1) to give compound 2 as a colorless oil (670 mg, 80%). [M+H]⁺=696.9

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carboxylate (3)

A solution of 2 (670 mg, 0.96 mmol) in CHOOH (1.5 mL) was treated with an aqueous solution of formaldehyde (37% in water, 0.77 ml, 1.15 mmol) and allowed to stir at 50° C. for 1 h. After this time the reaction mixture was purified with DCM/MeOH/AcOH=100/1/0.5% to give 3 (500 mg, 73%) as a colorless oil. [M+H]⁺=710.9

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)-4-methylpiperazine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)phenoxy)acetic acid (Rae17)

A solution of 9 (0.5 g, 0.7 mmol) in HCOOH (40 mL) was heated to 40° C. for 2 h. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae17 (368.7 mg, 80%) as a white solid.

FKBD Example 32 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-4-fluorophenoxy)acetic acid (Rae18)

(E)-3-(3,4-dimethoxyphenyl)-1-(2-fluoro-5-hydroxyphenyl)prop-2-en-1-one (4). A (3)

To the solution of 3,4-dimethoxybenzaldehyde 1 (5 g, 30.129.8 mmol) and 1-(2-fluoro-5-% Pd/C (2 g) in THF (200 m) was hydrogenated with hydroxyphenyl)ethan-1-one 2 (4.6 g, 30.1 mmol) in EtOH (60 mL) was added a solution of 10% aqueous NaOH (50 mL, 120.4 mmol) at 0° C. The resulting solution was stirred at room temperature for 12 h. The solution was adjusted to pH 4 by added 4M aqueous HCl dropwise at 0° C., generated a large of yellow solid. Then the mixture was filtered and the solid was washed with water (50 mL) to afford 3 (9 g, 99%) as a yellow solid. [M+H]⁺=303.2

3-(3,4-dimethoxyphenyl)-1-(2-fluoro-5-hydroxyphenyl)propan-1-one (4)

A solution of 3 (9 g, 29.8 mmol) and 10% Pd/C (2 g) in THF (200 mL) was hydrogenated with H₂ for 12 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product was used to the next step without any further purification. [M+H]⁺=304.8

tert-butyl 2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)-4-fluorophenoxy)acetate (5)

A solution of 4 (10 g, 32.8 mmol) and K₂CO₃ (9 g, 65.6 mmol) in DMF (200 mL) was treated with tert-butyl bromoacetate (7.7 g, 39.3 mmol) and allowed to stir at room temperature for 8 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated. The mixture was filtered and the solid was washed with water (300 mL). The crude product was purified by column chromatography on silica gel (AcOEt/PE 1:6) to give 5 (4 g, 32%, 2 steps) as a yellow oil. [M+Na]⁺=441.0

tert-butyl (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-4-fluorophenoxy)acetate (6)

A solution of ketone 5 (4 g, 9.56 mmol) in dry THF (30 mL) at −20° C. was treated with a solution of (+)-DIPChloride (28.68 mmol) in heptane (1.7 M, 16.8 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 6, then quenched with 2,2′-(ethylenedioxy)diethylamine (4.2 g) by forming an insoluble complex. After stirring at room temperature for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 6 (2 g, 50%, ee 93%) as a light yellow oil. [M+Na]⁺=442.7

(R)-1-(5-(2-(tert-butoxy)-2-oxoethoxy)-2-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

A solution of 6 (2 g, 4.76 mmol) and 7 (2.22 g, 7.14 mmol) in CH₂Cl₂(20 mL) was cooled to −20° C. before a solution of DCC (1.47 g, 7.14 mmol) in CH₂Cl₂ (10 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (60 mg, 0.47 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:2) to give compound 8 (1.8 g, 45%) as a light yellow oil. [M+Na]⁺=736.3

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-4-fluorophenoxy)acetic acid (Rae18)

A solution of 8 (1.7 g, 2.52 mmol) in CH₂Cl₂ (10 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (10 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:3:0.5%) to afford Rae18 (705 mg, 42%) as a white solid.

FKBD Example 33 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-5-fluorophenoxy)acetic acid (Rae-19)

(E)-3-(3,4-dimethoxyphenyl)-1-(3-fluoro-5-hydroxyphenyl)prop-2-en-1-one (3)

To the solution of 3,4-dimethoxybenzaldehyde 1 (6.391 g, 38.5 mmol) and 1-(3-fluoro-5-hydroxyphenyl)ethan-1-one 2 (5.39 g, 35 mmol) in EtOH (70 m) was added a solution of 40% aqueous KOH (19.6 g, 140 mmol) at 0° C. The resulting solution was reacted at room temperature for 4 h. The yellow solid was filtrated to give compound 3 (8.3 g, 78%). [M+H]⁺=303.0

tert-butyl (E)-2-(3-(3-(3,4-dimethoxyphenyl)acryloyl)-5-fluorophenoxy)acetate (4)

A solution of 3 (8.3 g, 27.5 mmol) and K₂CO₃ (4.55 g, 32.9 mmol) in DMF (80 mL) was treated with tert-butyl bromoacetate (6.4 g, 32.9 mmol) and allowed to stir at room temperature for 4 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were concentrated in vacuo, which was used for the next step without purification (11.11 g, 97%). [M+Na]⁺=439.2

tert-butyl 2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)-5-fluorophenoxy)acetate (5)

A solution of 4 (11.11 g, 26.7 mmol) and 10% Pd/C (1.11 g) in THF (200 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 5 as a colorless oil (4.2 g, 38%). [M+Na]⁺=440.7

tert-butyl (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-5-fluorophenoxy)acetate (6)

A solution of ketone 5 (4.2 g, 10 mmol) in dry THF (40 mL) at −20° C. was treated with a solution of (+)-DIPChloride (20 mmol) in heptane (1.7 M, 11.8 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 5, then quenched with 2,2′-(ethylenedioxy)diethylamine (2.9 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 6 as a light yellow oil (2.94 g, 70%, ee 98% vs racemate). [M+Na]⁺=443.0

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)-5-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

A solution of 6 (1.8 g, 4.28 mmol) and 7 (2 g, 6.42 mmol) in CH₂Cl₂ (18 mL) was cooled to −20° C. before a solution of DCC (1.33 g, 6.42 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 52 mg, 0.43 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 8 as a light yellow oil (2.7 g, 90%). [M+Na]⁺=735.9

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-5-fluorophenoxy)acetic acid (Rae19)

A solution of 8 (2.7 g, 4.11 mmol) in CH₂Cl₂ (12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae19 (1.094 g, 44%) as a pale yellow solid.

FKBD Example 34 2-(5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-fluorophenoxy)acetic acid (Rae20)

(E)-3-(3,4-dimethoxyphenyl)-1-(4-fluoro-3-hydroxyphenyl)prop-2-en-1-one (3)

To the solution of 3,4-dimethoxybenzaldehyde 1 (6.391 g, 38.5 mmol) and 1-(4-fluoro-5-hydroxyphenyl)ethan-1-one 2 (5.39 g, 35 mmol) in EtOH (70 mL) was added a solution of 40% aqueous KOH (19.6 g, 140 mmol) at 0° C. The resulting solution was reacted at room temperature for 4 h. The yellow solid was filtrated to give compound 3 (9.368 g, 89%). [M+H]⁺=303.2

tert-butyl (E)-2-(5-(3-(3,4-dimethoxyphenyl)acryloyl)-2-fluorophenoxy)acetate (4)

A solution of 3 (9.368 g, 31 mmol) and K₂CO₃ (5.1 g, 37 mmol) in DMF (90 mL) was treated with tert-butyl bromoacetate (7.2 g, 37 mmol) and allowed to stir at room temperature for 4 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were concentrated in vacuo, which was used for the next step without purification (13 g, quant.). [M+Na]⁺=438.9

tert-butyl 2-(5-(3-(3,4-dimethoxyphenyl)propanoyl)-2-fluorophenoxy)acetate (5)

A solution of 4 (13 g, 31.2 mmol) and 10% Pd/C (1.3 g) in THF (200 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 5 as a colorless oil (7 g, 54%). [M+Na]⁺=441.2

tert-butyl (R)-2-(5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-2-fluorophenoxy)acetate (6)

A solution of ketone 5 (7 g, 16.7 mmol) in dry THF (40 mL) at −20° C. was treated with a solution of (+)-DIPChloride (33.5 mmol) in heptane (1.7 M, 19.7 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 5, then quenched with 2,2′-(ethylenedioxy)diethylamine (4.89 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 6 as a light yellow oil (4.9 g, 71%, ee 96% vs racemate). [M+Na]⁺=443.3

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)-4-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

A solution of 6 (1.8 g, 4.28 mmol) and 7 (2 g, 6.42 mmol) in CH₂Cl₂ (18 mL) was cooled to −20° C. before a solution of DCC (1.33 g, 6.42 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 52 mg, 0.43 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 8 as a light yellow oil (2 g, 65%). [M+Na]⁺=736.4

2-(5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-fluorophenoxy)acetic acid (Rae20)

A solution of 8 (1.8 g, 2.52 mmol) in CH₂Cl₂ (12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae20 (835 g, 50%) as a pale yellow solid.

FKBD Example 35 2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-fluorophenoxy)acetic acid (Rae21)

(E)-3-(3,4-dimethoxyphenyl)-1-(2-fluoro-3-hydroxyphenyl)prop-2-en-1-one (3)

To the solution of 3,4-dimethoxybenzaldehyde 1 (9.7 g, 58.4 mmol) and 1-(2-fluoro-3-=303.0

tert-butyl (E)-2-(3-(3-(3,4-dimethoxyphenyl)acryloyl)-2-fluorophenoxy)acetate (4)

A solution of 3 (3.5 g, 11.6 mmol) and K₂CO₃ (1.92 g, 13.9 mmol) in DMF (40 mL) was treated with tert-butyl bromoacetate (2.7 g, 13.9 mmol) and allowed to stir at room temperature for 4 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The combined organic layers were concentrated in vacuo, which was used for the next step without purification (3.9 g, 80%). [M+H]⁺=416.9

tert-butyl 2-(3-(3-(3,4-dimethoxyphenyl)propanoyl)-2-fluorophenoxy)acetate (5)

A solution of 4 (3.5 g, 8.4 mmol) and 10% Pd/C (350 mg) in THF (50 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:1) to give compound 5 as a colorless oil (2.45 g, 70%). [M+Na]⁺=441.0

tert-butyl (R)-2-(3-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)-2-fluorophenoxy)acetate (6)

A solution of ketone 5 (2.45 g, 5.85 mmol) in dry THF (30 mL) at −20° C. was treated with a solution of (+)-DIPChloride (17.6 mmol) in heptane (1.7 M, 10.3 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 5, then quenched with 2,2′-(ethylenedioxy)diethylamine (3 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 6 as a light yellow oil (2.3 g, 94%, ee>99%). [M+Na]⁺=443.0

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)-2-fluorophenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

A solution of 6 (1.728 g, 4.1 mmol) and 7 (1.919 g, 6.15 mmol) in CH₂Cl₂ (18 mL) was cooled to −20° C. before a solution of DCC (1.26 g, 6.15 mmol) in CH₂Cl₂ (5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 49 mg, 0.4 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 8 as a light yellow oil (2 g, 70%). [M+Na]⁺=735.7

2-(3-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-fluorophenoxy)acetic acid (Rae21)

A solution of 8 (2 g, 2.52 mmol) in CH₂Cl₂(12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae21 (1.238 g, 67%) as a white solid.

FKBD Example 36 2-(5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-hydroxyphenoxy)acetic acid (Rae24)

1-(4-(benzyloxy)-3-hydroxyphenyl)ethan-1-one (2)

A solution of 1 (19 g, 125 mmol) and K₂CO₃ (17.2 g, 125 mmol) in DMF (250 mL) was treated with benzyl bromide (21.2 g, 125 mmol) and allowed to stir at room temperature for 12 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated. The mixture was filtrated and the solid was washed with water (300 mL) to give 2 (13 g, 43%) as a white solid. [M+H]⁺=243.1

(E)-1-(4-(benzyloxy)-3-hydroxyphenyl)-3-(3,4-dimethoxyphenyl)prop-2-en-1-one (4)

To the solution of 2 (12.7 g, 52.47 mmol) and 3 (10.5 g, 62.97 mmol) in EtOH (60 mL) was added a solution of 40% aqueous KOH (8.4 g, 209.8 mmol) at 25° C. The resulting solution was heated to 45° C. for 8 h. The solution was adjusted to pH 4 by added 4M aqueous HCl dropwise at 0° C., generated a large of yellow solid. Then the mixture was filtered and the filter cake was washed with water (100 mL) to afford 4 (16.5 g, 80%) as a yellow solid. [M+H]⁺=391.2.

tert-butyl (E)-2-(2-(benzyloxy)-5-(3-(3,4-dimethoxyphenyl)acryloyl)phenoxy)acetate (5)

A solution of 4 (16.4 g, 42 mmol) and K₂CO₃ (11.6 g, 84.1 mmol) in DMF (50 mL) was treated with tert-butyl bromoacetate (12.23 g, 63.07 mmol) and allowed to stir at room temperature for 12 h. After this time the reaction mixture was poured into ice, yellow solid was precipitated. The mixture was filtered and the solid was washed with water (100 mL). The crude product was washed by petroleum ether (100 mL) to give 5 (18.5 g, 88%) as a yellow solid. [M+H]⁺=504.9.

tert-butyl 2-(5-(3-(3,4-dimethoxyphenyl)propanoyl)-2-hydroxyphenoxy)acetate (6)

A solution of 5 (18.0 g, 35.7 mmol) and 10% Pd/C (2 g) in THF (400 mL) was hydrogenated with H₂ for 4 h at room temperature. The reaction mixture was then filtered and concentrated. The crude product 6 (16 g, 88%) was used to the next step directly. [M+Na]⁺=439.0

tert-butyl 2-(2-((tert-butoxycarbonyl)oxy)-5-(3-(3,4-dimethoxyphenyl)propanoyl)phenoxy)acetate (7)

A solution of 6 (3 g, 7.2 mmol) and Boc₂O (2.35 g, 10.8 mmol) in dry DCM (60 mL) at 25° C. was treated with DMAP (0.87 g, 7.2 mmol) at 25° C. After stirring at room temperature for 1 h, the solution was concentrated in vacuum. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 7 (2.5 g, 67%) as a light yellow oil. [M+Na]⁺=538.9.

tert-butyl (R)-2-(2-((tert-butoxycarbonyl)oxy)-5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)phenoxy)acetate (8)

A solution of 7 (2.3 g, 4.45 mmol) in dry THF (20 mL) at −20° C. was treated with a solution of (+)-DIPChloride (13.3 mmol) in heptane (1.7 M, 8 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 7, then quenched with 2,2′-(ethylenedioxy)diethylamine (1.97 g) by forming an insoluble complex. After stirring at room temperature for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:4) to give compound 8 (2 g, 86%) as a light yellow oil. [M+Na]⁺=540.9.

(R)-1-(3-(2-(tert-butoxy)-2-oxoethoxy)-4-((tert-butoxycarbonyl)oxy)phenyl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (10)

A solution of 8 (2 g, 3.86 mmol) and 9 (1.8 g, 5.79 mmol) in CH₂Cl₂ (15 mL) was cooled to −20° C. before a solution of DCC (1.19 g, 5.79 mmol) in CH₂Cl₂(5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (47 mg, 0.38 mmol) in CH₂Cl₂(2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 10 (2.2 g, 70%) as a light yellow oil. [M+Na]⁺=833.8.

2-(5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)-2-hydroxyphenoxy)acetic acid (Rae24). Condition 1

A solution of 10 (50 mg, 0.06 mmol) in CH₂Cl₂ (2 mL) was treated with a solution of 20% TFA in CH₂Cl₂ (1 mL) at 0° C. The mixture stirred at room temperature for 1 h. LCMS analysis showed no desired product and start material can be detected. Condition 2: A solution of 10 (50 mg, 0.06 mmol) in HCOOH (1 mL) was stirred at room temperature for 1 h. LCMS analysis showed no desired product and start material can be detected.

FKBD Example 37 2-((5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-yl)oxy)acetic acid (Rae26)

(E)-3-(3,4-dimethoxyphenyl)-1-(5-hydroxypyridin-3-yl)prop-2-en-1-one (3)

To the solution of 3,4-dimethoxybenzaldehyde 1 (5.0 g, 30.1 mmol) and 1-(5-hydroxypyridin-3-yl)ethan-1-one 2 (4.95 g, 36.12 mmol) in EtOH (200 mL) was added a solution of 40% aqueous KOH (16.83 g, 120 mmol) at 0° C. The resulting solution was reacted at room temperature for 8 h, followed by dilution with EtOAc. The organic layer was washed by water, brine, dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:3) to give compound 3 as a colorless oil (6.8 g, 80%). [M+H]⁺=285.9

tert-butyl (E)-2-((5-(3-(3,4-dimethoxyphenyl)acryloyl)pyridin-3-yl)oxy)acetate (4)

A solution of 3 (6 g, 21.03 mmol) and K₂CO₃ (3.5 g, 25.24 mmol) in DMF (150 mL) was treated with tert-butyl bromoacetate (4.93 g, 25.24 mmol) and allowed to stir at room temperature for 4 h. After this time the reaction mixture was quenched by H₂O and extracted with EtOAc twice. The organic layers were dried over Na₂SO₄ and concentrated in vacuo. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 4 as a yellow oil (4.5 g, 54%). [M+H]⁺=399.9

tert-butyl 2-((5-(3-(3,4-dimethoxyphenyl)propanoyl)pyridin-3-yl)oxy)acetate (5)

A solution of 4 (4.5 g, 11.26 mmol) and 10% Pd/C (400 mg) in THF (100 mL) was hydrogenated with H₂ for 6 h at room temperature. The reaction mixture was then filtered and concentrated. The residue was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 5 as a yellow oil (2.5 g, 56%) [M+H]⁺=402.2

tert-butyl (R)-2-((5-(3-(3,4-dimethoxyphenyl)-1-hydroxypropyl)pyridin-3-yl)oxy)acetate (6)

A solution of ketone 5 (2.5 g, 6.23 mmol) in dry THF (40 mL) at −20° C. was treated with a solution of (+)-DIPChloride (24.9 mmol) in heptane (1.7 M, 14.7 mL) at −20° C. The resulting mixture was reacted at −20° C. until complete conversion of 5, then quenched with 2,2′-(ethylenedioxy)diethylamine (3.7 mL) by forming an insoluble complex. After stirring at RT for another 30 min, the suspension was filtered through a pad of celite and concentrated. The crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:5) to give compound 6 as a colorless oil (2 g, 80%, ee>99%). [M+H]⁺=404.0

(R)-1-(5-(2-(tert-butoxy)-2-oxoethoxy)pyridin-3-yl)-3-(3,4-dimethoxyphenyl)propyl (S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carboxylate (8)

A solution of 6 (1.898 g, 4.7 mmol) and 7 (2.196 g, 7.1 mmol) in CH₂Cl₂ (18 mL) was cooled to −20° C. before a solution of DCC (1.46 g, 7.1 mmol) in CH₂Cl₂(5 mL) was added, followed by the addition of a solution of 4-(dimethylamino)pyridine (DMAP, 61 mg, 0.5 mmol) in CH₂Cl₂ (2 mL) under argon atmosphere. The resulting white suspension was allowed to stir at −20° C. for 2 h. The reaction mixture was then filtered, evaporated, and the crude compound was purified by silica-gel flash column chromatography (AcOEt/PE 1:7) to give compound 8 as a light yellow oil (2.05 g, 63%). [M+H]⁺=696.8

2-((5-((R)-1-(((S)-1-(4-(acryloyloxy)-3,3-dimethyl-2-oxobutanoyl)piperidine-2-carbonyl)oxy)-3-(3,4-dimethoxyphenyl)propyl)pyridin-3-yl)oxy)acetic acid (Rae26)

A solution of 8 (2 g, 2.87 mmol) in CH₂Cl₂ (12 mL) was treated with a solution of 40% TFA in CH₂Cl₂ (12 mL) at 0° C. The mixture was allowed to react at room temperature until complete conversion. The reaction mixture was charged to silica-gel flash column directly (AcOEt/PE/AcOH 1:2:0.5%) to afford Rae26 (545.8 g, 30%) as a white solid.

Linker Example 1

(Z)-hex-3-ene-1,6-diol (1)

Hex-3-yne-1,6-diol (2.0 g), quinoline (0.12 g) and Lindlar catalyst (0.30 g) were suspended in MeOH (15 mL). Hydrogen was filled in to the flask with a Schlenk line and a positive pressure was maintained with a balloon of hydrogen. The reaction was stirred at RT for 12 h before filtered and concentrated. The crude product (2.1 g) was co-evaporated with toluene (20 mL×2) to remove the residue of MeOH. The product 1 was used without further purification.

(Z)-6-hydroxyhex-3-en-1-yl 4-methylbenzenesulfonate (2)

Monotosylation of diol was obtained by a reported Ag₂O-assisted method (10). The percentage yield of monotosylation is 90% for cis-C6 linker on 2.0 g scale. ¹H NMR (500 MHz, CDCl₃) δ 7.79 (d, J=8.3 Hz, 2H, aromatic), 7.35 (d, J=8.0 Hz, 2H, aromatic), 5.63-5.48 (m, 1H, ═CH), 5.48-5.33 (m, 1H, ═CH), 4.04 (t, J=6.7 Hz, 2H, OCH2), 3.64 (dd, J=12.3, 6.2 Hz, 2H, OCH2), 2.45 (s, 3H, CH3), 2.44 (q, J=6.5 Hz, 2H), 2.28 (q, J=6.5 Hz, 2H). ¹³C NMR (126 MHz, CDCl₃) δ 129.86 (aromatic), 129.51 (aromatic), 127.93 (═CH), 126.19 (═CH), 69.66 (OCH2), 61.99 (OCH2), 30.89, 27.26, 21.69 (CH3). HRMS for [M+H]⁺ C13H18O4S, calculated: 271.1004, observed: 271.1004.

3

To conjugate the Ts-protected alcohol on 2-chlorotrityl chloride solid support, briefly, the resin (9.6 mmol, 1.14 mmol/g), 2,6-di-tert-butylpyridine (10.5 mmol) and alcohol (5.9 mmol) was mixed in 100 mL CH₂Cl₂. AgOTf (10.0 mmol) was added in two aliquots over 15 min. The red color of the resin persisted and this indicates that the alcohol is depleted in the reaction mixture. MeOH (5 mL) was then added to quench the reaction and the color turned white or pale yellow over 5 min. The suspension was stirred at RT for another 1 h before it was filtered and the solid-support was transferred to a separatory funnel with CCl₄. After the mixture standing for 5 min to allow stratification, AgCl precipitation on the bottom was removed by draining the liquid to a level that most floating resin remained. The resin was then collected in a 250 mL solid-support reactor and washed with pyridine (50 mL×4) with extensive shaking.

(Cis-C6 Linker).

The resin was then transferred into a 250 mL RB-flask with 100 mL THF. Methylamine (33% in MeOH) was added and stirred at 40° C. for 12 h. The resin was filtered and washed with THF (50 mL) for twice and CH₂Cl₂ (50 mL) for twice. For long time storage at −20° C., the resin was further washed with MeOH and air-dried for 20 min. The molarity of the NH group was determined by UV of the cleaved first coupled Fmoc group (0.40-0.45 mmol/g).

RAPAFUCIN EXAMPLES

General Automated Synthesis. Solid-phase peptide synthesis (SPPS) were applied with a split-pool strategy to assemble the tetrapeptide effector domains. The pre-assembled FKBD capped with a carboxylic acid at one end and an olefin at the other was subsequently coupled to the tetrapeptide that remained tethered on beads. To facilitate purification of the newly formed macrocycles, we adopted a coupled macrocyclization and cyclative release strategy whereby the macrocyclization is accompanied by the concurrent release of the macrocyclic products from the solid beads. After exploring different macrocyclization methods, ring-closing metathesis/cyclative release (RCM) can be used for efficient parallel synthesis of different Rapafucins. Both aFKBD and eFKBD possess high affinity for FKBP12, with K_(d) values of 4 and 11 nM, respectively. Importantly, this enhanced affinity was largely retained on incorporation into macrocycles, with average K_(d) values of 25 and 37 nM, respectively. Moreover, there was relatively low variation in binding affinity for FKBP12 among different macrocycles bearing aFKBD or eFKBD. These results suggested that both aFKBD and eFKBD are tolerant to different effector domain sequences, thus rendering them suitable FKBD building blocks for Rapafucin libraries.

Charged resin (4.800 g) was dissolved in DMF/DCM (1/4, v/v) and dispersed to each well of an Aapptec Vantage automated synthesizer (96 wells). Wells were drained and swelled with DMF for 20 mins before the solvent was drained and washed with 1×DMF. Fmoc-protected amino acid building blocks (3.0 eq., ˜0.3M in DMF), HATU (3.0 eq., ˜0.1M in DMF), and DIEA (6 eq., ˜0.3M in DMF) were added in order to each of the 96 wells. The resin and reagent mixture were mixed on the automated synthesizer for 2-3 hrs, then washed with DMF (5×) for 5 times. If coupling was difficult, the coupling reaction would be repeated. Resins were washed thoroughly with DMF (3×) for 3 times. Deprotection of the Fmoc group was achieved by shaking resins with 1 mL of piperidine/DMF (1/4, v/v) for 10 min and 1 mL piperidine/DMF (1/4, v/v) for 5 min. Resins were washed thoroughly with DMF 5 times. Coupling reaction was repeated 4 times to achieve the synthesis of tetrapeptide. Coupling reactions were repeated if Fmoc-valine or -isoleucine were to be coupled to N-methyl amino acids on resin or if Fmoc-proline was used. Then the deprotection of Fmoc group is performed. FKBD (3 eq., ˜0.2 M in DMF), HATU (3 eq., ˜0.1M in DMF), and DIEA (6 eq., ˜0.3M in DMF) were added in order into the vessel of the prepared resin. The resin and reagent mixture were mixed on the automated synthesizer for 3 hrs, then washed with DMF (2×) for 2 times and DCM (2×) for 2 times. 1.25 mL of Ethyl Acetate and 0.25 mL of Hoveyda-Grubbs II (30 mol %) were added to each well. The reaction block was 80° C. for 5 hrs. Upon reaction completion, the resulting brown suspension was purified on 1 g solid phase extraction columns packed with 1 g silica gel. The columns were washed using dichloromethane and eluted with 10% methanol in dichloromethane. The eluate was concentrated under vacuum and weighted. The compounds were characterized using LC/MS analysis.

TABLE 9 Synthesis and characterization of compounds 1066, 1081, 1082, 1087, 1088, and 1522. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Retention Uptake, No. monomer3/monomer4) weight time 293T Molecular Structure 1087 aFKBD ra602 ra140 dp ml 1289.54 3.92 low

1088 aFKBD ra348 mf dp ml 1276.54 4.11 low

1081 aFKBD ra602 ra553 dp ml 1338.61 4.24 medium

1082 aFKBD ra602 ra73 dp ml 1330.59 4.22 low

1522 aFKBD ra602 y dp ml 1240.46 3.65 high

1066 aFKBD ra602 ra559 dp ml 1262.51 4.07 high

General Manual Synthesis. Synthesized as previously described. (Guo et al. (2018) Nat. Chem. 11:254-63).

TABLE 10 Synthesis and characterization of compounds 560-574, 576, and 1563-65. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Retention Prolif, No. monomer3/monomer4) weight time A549 Chemical Structure 560 rae1 ra147 napA ra562 g 1247.49 5.56 medium

561 rae2 ra147 napA ra562 g 1247.49 5.63 medium

562 rae3 ra147 napA ra562 g 1247.49 5.48 medium

563 rae4 ra147 napA ra562 g 1247.49 5.47 low

564 rae5 ra147 napA ra562 g 1247.49 5.48 low

565 rae9 ra147 napA ra562 g 1233.47 5.35 medium

566 rae10 ra147 napA ra562 g 1233.47 5.10 medium

567 rae11 ra147 napA ra562 g 1233.47 5.11 medium

568 rae12 ra147 napA ra562 g 1235.46 5.74 medium

569 rae13 ra147 napA ra562 g 1235.46 5.27 medium

570 rae14 ra147 napA ra562 g 1235.46 5.72 medium

571 rae16 ra147 napA ra562 g 1440.70 5.93 low

572 rae17 ra147 napA ra562 g 1232.48 4.41 medium

573 rae18 ra147 napA ra562 g 1235.46 5.49 low

574 rae19 ra147 napA ra562 g 1235.46 5.60 low

576 rae20 ra147 napA ra562 g 1235.46 5.56 medium

1563 rae21 ra147 napA ra562 g 1235.46 6.94 high

1564 rae29 ra147 napA ra562 g 1204.44 6.67 high

1565 rae26 ra147 napA ra562 g 1218.46 low

TABLE 11 Synthesis and characterization of compounds 1566-84. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Prolif, No. monomer3/monomer4) weight H929 Chemical Structure 1566 rae1 my df sar df 1251.44 medium

1567 rae10 my df sar df 1237.41 medium

1568 rae11 my df sar df 1237.41 low

1569 rae12 my df sar df 1239.41 low

1570 rae13 my df sar df 1239.41 medium

1571 rae14 my df sar df 1239.41 low

1572 rae16 my df sar df 1444.65 low

1573 rae16a my df sar df 1222.40 low

1574 rae17 my df sar df 1236.43 low

1575 rae18 my df sar df 1239.41 low

1576 rae19 my df sar df 1239.41 medium

1577 rae2 my df sar df 1251.44 medium

1578 rae20 my df sar df 1239.41 low

1579 rae21 my df sar df 1239.41 medium

1580 rae26 my df sar df 1222.40 low

1581 rae3 my df sar df 1251.44 medium

1582 rae4 my df sar df 1251.44 low

1583 rae5 my df sar df 1251.44 low

1584 rae9 my df sar df 1237.41 low

TABLE 12 Synthesis and characterization of compounds 1555-1557. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Retention Uptake, No. monomer3/monomer4) weight time 293T Chemical Structure 1555 raa18 ra602 mf dp ml 1237.51 4.39 high

1556 rae27 ra602 mf dp ml 1211.46 5.02 low

1557 raa17 ra602 mf dp ml 1237.51 4.37 high

Post cyclization modification. Protecting groups may be removed before final purification. In some embodiments, a tert-butyl protecting group can be removed using TFA. A solution of protected Rapafucin is dissolved in DCM and triethylsilane (2 Eq) is added. TFA (20% final concentration) is added and stirred for 2 hours. The mixture is reduced under vacuum and purified via normal phase chromatography (1:9 MeOH/DCM) to give a yellow solid. The compound is further reunified using reverse phase chromatography (40-95% ACN/H₂O) to give a pale colored solid.

In some embodiments, a tert-butyloxycarbonyl protecting group may be removed using TFA. A solution of protected Rapafucin is dissolved in DCM and triethylsilane (2 Eq) is added. TFA (20% final concentration) is added and stirred for 2 hours. The mixture is reduced under vacuum and purified via normal phase chromatography (1:9 MeOH/DCM) to give a yellow solid. The compound is further reunified using reverse phase chromatography (40→95% ACN/H₂O) to give a pale colored solid.

Additional functional groups can be added to deprotected Rapafucins. In some embodiments, reactive functional groups can be deprotected to produce a chemical handle for additional modifications. These reactions include substitution, addition, and radical reactions.

In some embodiments, a carbamate group is appended to an alcohol containing rapafucin. Other functional groups would work as well. This is an example of attaching an electrophile to the exposed nucleophile, in this embodiment, a phenol group. A deprotected alcohol (or phenol) containing Rapafucin is dissolved in DCM, then pyridine (10 mol %) and DIEA (3 Eq) was added. A solution of carbonyl chloride (3 Eq) in DCM was added dropwise and stirred for 2 hours. The solution was washed with a saturated ammonium chloride solution (3×) and dried over Mg₂SO₄. The solution concentrated and purified via column chromatography (0→20% MeOH/EtOAc) to produce a white solid.

TABLE 13 Synthesis and characterization of compounds 867-869 and 877. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Retention Prolif. No. monomer3/monomer4) weight time H929 Chemical Structure 877 rae37 ra398 df sar df 1319.52 4.181 low

867 rae21 ra492 df sar df 1352.52 5.75 low

868 rae19 ra492 df sar df 1352.52 5.54 low

869 aFKBD ra492 df sar df 1375.58 5.403 low

In some embodiments, an amide group is formed from an amine containing Rapafucin. A deprotected amine containing Rapafucin is dissolved in DCM, then acyl chloride (2 Eq) and DIEA (3 Eq) was added. The solution was washed with brine (3×) and dried over Mg₂SO₄. The solution concentrated and purified via column chromatography (0→20% MeOH/EtOAc) to produce a white solid.

TABLE 14 Synthesis and characterization of compounds 1585-1589. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Retention Uptake, No. monomer3/monomer4) weight time 293T Chemical Structure 1585 afkbd phg ra655 dp ml 1357.60 3.72 High

1586 afkbd phg ra656 dp ml 1370.70 3.74 Med

1587 afkbd phg ra626 dp ml 1338.60 3.15 Low

1588 afkbd phg ra592 dp ml 1281.52 3.44 High

1589 afkbd phg ra618 dp ml 1358.60 3.10 Low

In some embodiments, an amide group is formed from carboxylic acid containing rapafucin. A deprotected carboxylic acid containing Rapafucin is dissolved in ethyl acetate (5 mM), then an amine (2 Eq), DIEA (10 Eq), and T3P (2 Eq) was added. The reaction until the reaction was complete via LC/MS. The solution was washed with brine (3×) and the organic layer was dried over Mg₂SO₄. The solution concentrated and purified via column chromatography (0→20% MeOH/W EtOAc) to produce a white solid.

TABLE 15 Synthesis and characterization of compounds 1558, 1559, 1562, 1590, and 1591. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Retention Uptake, No. monomer3/monomer4) weight time 293T Chemical structure 1558 afkbd phg ra500 dp ml 1311.50 3.81 high

1559 afkbd phg ra501 dp ml 1343.60 3.86 medium

1562 afkbd phg ra504 dp ml 1344.60 3.22 low

1590 afkbd phg ra620 dp ml 1371.64 3.919 Low

1591 afkbd phg ra623 dp ml 1365.68 3.956 Low

In some embodiments, a phosphinate group may be added to a rapafucin. A deprotected alcohol (or phenol) containing Rapafucin is dissolved in DCM and pyridine (1:1 v/v) and dimethylphosphinic chloride (11 Eq) at room temperature and stirred for 16 hrs. The reaction mixture was diluted with DCM and washed with dilute HCl. The organic fraction was washed with water and dried over Mg₂SO₄. The solution concentrated and purified via column chromatography (0→20% MeOH/W EtOAc) to produce a white solid.

TABLE 16 Synthesis and characterization of compound 1520. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Retention Uptake, No. monomer3/monomer4) weight time 293T Chemical structure 1520 aFKBD ra602 ra515 dp ml 1316.4 5.34 low

Manual Gram Scale Ring-Closing Metathesis. Charged Resin (Loading Capacity=0.2-0.3 mmol/g) is loaded in a 500 ml of SPPS vessel and swelled for 30 min with DCM (300 ml) on laboratory shaker (Kamush® LP360AMP, 360°, speed 6), then filtered and washed with DMF (200 ml×2) and dried under vacuum for 5 min.

A solution of Fmoc-AA (3 eq) and HATU (3 eq) in 150 ml of DMF was added to the resin. Then DIEA (6 eq) in 50 ml of DMF was added and shaken for 3 hrs. Solvent was filtered and washed with DMF (200 ml×5) and DCM (200 ml×5) and dried. 300 ml of 20% Piperidine in DMF was added and shaken for 20-30 min, filtered and again 300 ml of 20% Piperidine in DMF was added and shaken for 20-30 min. The solvent was filtered and washed carefully with DMF (200 ml×5), then immediately taken for next Fmoc-AA coupling.

After the peptidic portion is installed and deprotected, FKBD (2 eq) was also coupled similar manner was taken for next step (No de-protection of the FKBD necessary). LC-MS analysis was performed after every Fmoc-AA coupling.

Linear Rapafucin on resin and Hoveyda-Grubbs II (30 mol %) was taken in a 2 L round bottom flask with 8 cm long octagonal stir bar. Ethyl acetate (600 mL) was taken in 2 L conical flask and sparged with gentle stream of N₂ for −10 min, then was added to the Resin/Catalyst mixture. A super air condenser was mounted and the flask was placed in oil bath and heated to 90° C. for 5 h (moderate reflux) under N₂ (Balloon). The solution was cooled to room temperature leaving a dark brown solution with suspended resin. The resin was checked using LC/MS and TLC for formation of desired product.

Resin was filtered off and the filtrate was evaporated in vacuo to generate a dark brown crude product which was dissolved in minimal DCM (60 mL) and subjected into normal phase column chromatography (0→10% MeOH/EtOAc). Fractions containing pure desired compound were pooled and concentrated in vacuo to yield a brownish powder. The product was then dissolved in a minimal amount of MeOH (20 mL) and subjected into reverse phase column chromatography (10 to 95% ACN/H₂O). Fractions containing pure desired compound were pooled and concentrated in vacuo to get off-white solid, which was dissolved in 20-25 ml of 2-MeTHF and dripped into the 250 ml of Heptane in a 1 L flask with gentle stirring. Formed white precipitate was filtered and dried to get pale grayish white powder.

TABLE 17 Synthesis and characterization of compound 1592. Composition (FKBD/ Compound monomer1/monomer2/ Molecular Retention A549 No. monomer3/monomer4) weight time Prolif Molecular Structure 1592 aFKBD ml df mi g 1178.44 6.48 High

Ring Closing via Macrolactamization. Unmodified 2-chloro-chlorotrityl resin (Loading Capacity=1.5 mmol/g) is loaded into a solid phase reaction vessel (60 mL) and peptidic portion is synthesized under normal solid phase synthesis conditions. (see above section).

For peptide residues that need alternative coupling conditions for racemization, the resin may be treated to the following conditions: Deprotected resin is cooled to 0° C. Resin was treated with a cold (0° C.) pre-mixed (5 minutes) solution of FMOC-Amino Acid (3 Eq) in DMF, Oxyma (3 Eq) in DMF and DIEA (3 Eq); shaken for 3 hours. The resultant resin was filtered and washed with DMF (5×3 ml), DCM (5×3 ml) and dried.

After deprotection of the peptidic portion on resin, a FKBD containing a protected amine functionality can be installed using normal synthetic procedures. The resultant fragment can be deprotected and released from the resin.

The FKBD containing linear rapafucin can be further cyclized to produce the cyclic Rapafucin. Acyclic Rapafucin is taken up in DMF and treated with COMU-PF6 (3 Eq) and DIEA (3 Eq), let stir for 1 hour. The reaction is monitored by LC/MS. Upon completion, the mixture is diluted with water and extracted with EtOAc (3×). Combined extracts were washed with brine, dried over MgSO₄ and reduced under vacuum. The crude product is purified via column chromatography (1:9 MeOH/EtOAc) to give an orange solid and repurified via reverse phase chromatography (40→95% ACN/H₂O) to give a tan solid.

If required protecting groups may be removed before final purification. In some embodiments, a tert-butyl protecting group can be removed using TFA. A solution of protected Rapafucin is dissolved in DCM and triethylsilane (2 Eq) is added. TFA (20% final concentration) is added and stirred for 2 hours. The mixture is reduced under vacuum and purified via normal phase chromatography (1:9 MeOH/DCM) to give a yellow solid. The compound is further reunified using reverse phase chromatography (40→95% ACN/H₂O) to give a pale colored solid.

TABLE 18 Synthesis and characterization of compound 1593. Composition (FKBD/ Compound monomer1/monomer2/ Retention Molecular Uptake, No. monomer3/monomer4) time weight 293T Chemical structure 1593 aFKBD ra602 Ra503 dp ml 5.09 1354.61 High

Prophetic Examples—DNA-Encoded Library Prophetic Example 1—Preparation of a Rapafucin DNA-Encoding Library Via Split-and-Pool Cycles

A rapafucin DNA-encoding library is synthesized by a sequence of split-and-pool cycles wherein the oligonucleotide is attached to the FKBD. First, an initial oligonucleotide of Formula (XIII) is synthesized and HPLC purified. A first building block comprising an FKBD building block is then covalently bound to the oligonucleotide of Formula (XIII) via click chemistry. Subsequently, a second oligonucleotide, encoding the first building block, is appended to the oligonucleotide of Formula (XIII). The resulting product is pooled and split into a second set of separate reaction vessels and a second building block comprising an effector domain building block is coupled to the first building block using a ring-closing reaction. The reaction is then encoded by the attachment of a unique oligonucleotide sequence to the unique oligonucleotide attached to the first building block. The encoded two-building-block molecules yields the final library.

Prophetic Example 2—Preparation of a Rapafucin DNA-Encoding Library Via Split-and-Pool Cycles

A rapafucin DNA-encoding library is synthesized by a sequence of split-and-pool cycles wherein the oligonucleotide is attached to a linking region. First, an initial oligonucleotide of Formula (XIII) is synthesized and HPLC purified. Then, the oligonucleotide of Formula (XIII) is covalently bound to a first linking region via click chemistry. A first building block comprising an FKBD building block is encoded by a second oligonucleotide which is appended to the initial oligonucleotide of Formula (XIII). The resulting product is pooled and split into a second set of separate reaction vessels and a second building block comprising an effector domain building block is coupled to the first building block using a ring-closing reaction. The reaction is then encoded by the attachment of a unique oligonucleotide sequence to the unique oligonucleotide attached to the first building block. The encoded two-building-block molecules yields the final library.

Prophetic Example 3—Preparation of a Rapafucin DNA-Encoding Library Via DNA-Recorded Synthesis and Ligation

A rapafucin DNA-encoding library is synthesized by DNA-recorded synthesis wherein the oligonucleotide is attached to the FKBD. First, an initial oligonucleotide of Formula (XIII) is synthesized and HPLC purified. A first building block comprising an FKBD building block is then covalently bound to the oligonucleotide of Formula (XIII) via click chemistry. Then, a second building block comprising an effector domain building block is coupled to the first building block via the first and second linking region through a ring-closing reaction. The reaction is encoded by DNA-recorded synthesis by ligation of a unique oligonucleotide to the initial oligonucleotide of formula (XIII).

Prophetic Example 4—Preparation of a Rapafucin DNA-Encoding Library Via DNA-Recorded Synthesis and Enzymatic Reactions

A rapafucin DNA-encoding library is synthesized by DNA-recorded synthesis wherein the oligonucleotide is attached to the FKBD. First, an initial oligonucleotide of Formula (XIII) is synthesized and HPLC purified. A first building block comprising an FKBD building block is then covalently bound to the oligonucleotide of Formula (XIII) via click chemistry. Then, a second building block comprising an effector domain building block is coupled to the first building block via the first and second linking region through a ring-closing reaction. The reaction is then encoded by DNA-recorded synthesis by polymerase-catalyzed fill-in reactions.

Prophetic Example 5—Preparation of a Rapafucin DNA-Encoding Library Via DNA-Templated Synthesis

A rapafucin DNA-encoding library is synthesized by DNA-templated synthesis. First, a second building block comprising an effector domain building block is coupled to the first building block comprising the FKBD via the first and second linking regions. Then, the reaction is encoded by DNA-templated synthesis, wherein a plurality of conjugate molecules of oligonucleotide-tagged building blocks are prepared and the spatial proximity of the two distinct oligonucleotides of Formula (XIII) facilitates the bimolecular chemical reactions between the two building blocks.

Examples—Biological Assays

Nucleoside Uptake Assay (uptake). Nuceloside uptake assays were performed with using 3H-Thymidine as described in Guo et al. (2018) Nat. Chem. 11:254-63. Specific cell lines are indicated in each assay and cultured in complete growth media. Activity is scored according to the IC₅₀ values relative to DMSO control. “Low” indicates an IC₅₀ greater than 600 nM, “Medium” indicates an IC₅₀ between 300 nM and 600 nM “High” indicates an IC₅₀ less than 300 nM. “Rel.Uptake” refers to uptake activity characterization relative to a single concentration assay. “Low” indicates a response greater than 0.6 times the activity relative to DMSO, “Medium” indicates a response between 0.6 and 0.3 times the activity relative to DMSO, “High” indicates a response less than 0.3 times the activity relative to DMSO.

Cell Proliferation Assay (Prolif.) Guo et al. (2018) Nat. Chem. 11:254-63. Specific cell lines are indicated in each assay and cultured in complete growth media. Activity is scored according to the IC₅₀ values relative to DMSO control. “Low” indicates an IC₅₀ greater than 600 nM, “Medium” indicates an IC₅₀ between 300 nM and 600 nM “High” indicates an IC₅₀ less than 300 nM. “Rel.Uptake” refers to uptake activity characterization relative to a single concentration assay. “Low” indicates a response greater than 0.6 times the activity relative to DMSO, “Medium” indicates a response between 0.6 and 0.3 times the activity relative to DMSO, “High” indicates a response less than 0.3 times the activity relative to DMSO.

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific composition and procedures described herein. Such equivalents are considered to be within the scope of this disclosure, and are covered by the following claims. 

What is claimed is:
 1. A macrocyclic compound, comprising: an FKBP-binding domain (FKBD); an effector domain; a first linking region; and a second linking region; wherein the FKBD, the effector domain, the first linking region, and the second linking region together form a macrocycle.
 2. A compound of Formula (V)

or an optically pure stereoisomer or pharmaceutically acceptable salt thereof, wherein L is selected from the groups in Table 1; A is CH₂, NH, NMe, O, S(O)₂ or S; each X is independently O, NMe, or NH; E is CH or N; each of R¹, R², R³, and R⁴ is independently selected from the group consisting of H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, OCF₃, OCHF₂, COC₁₋₂₀alkyl, CO₂C₁₋₂₀alkyl, C₃₋₈cycloalkyl, C₂₋₈alkenyl, C₂₋₈alkynyl, C₁₋₁₀alkoxy, C₆₋₁₅aryl, C₆₋₁₅aryloxy, C₆₋₁₅arylthio, C₂₋₁₀carboxyl, C₁₋₁₀alkylamino, thiol, C₁₋₁₀alkylthio, C₁₋₁₀alkyldisulfide, C₆₋₁₅arylthio, C₁₋₁₀heteroarylthio, (C₃₋₈cycloalkyl)thio, C₂₋₁₀heterocyclylthio, sulfonyl, C₁₋₁₀alkylsulfonyl, amido, C₁₋₁₀alkylamido, selenol, C₁₋₁₀alkylselenol, C₆₋₁₅arylselenol, C₁₋₁₀heteroarylselenol, (C₃₋₈cycloalkyl)selenol, C₂₋₁₀heterocyclylselenol, guanidino, C₁₋₁₀alkylguanidino, urea, C₁₋₁₀alkylurea, ammonium, C₁₋₁₀alkylammonium, cyano, C₁₋₁₀alkylcyano, C₁₋₁₀alkylnitro, adamantine, phosphonate, C₁₋₁₀alkylphosphonate, and C₆₋₁₅arylphosphonate, each of the above can be optionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₂₀alkyl, substituted C₁₋₂₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl; or R⁴ forms a cyclic structure formed with R³, the cyclic structure is selected from the group consisting of C₂₋₁₀heterocyclyl and C₁₋₁₀heteroaryloptionally substituted with H, halogen, hydroxyl, N₃, NH₂, NO₂, CF₃, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acyl C₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonyl C₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, substituted C₁₋₁₀alkylthio, and thiocarbonyl n is an integer selected from 0 to 4; m is an integer selected from 0 to 5; p is an integer selected from 0 to 2; q is an integer selected from 1 to 10;
 3. The compound of claim 2, wherein q is 3 or
 4. 4. A pharmaceutical composition comprising a compound of Formula (V) or an optically pure stereoisomer or pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.
 5. A method of making a Rapafucin macrocyclic molecule, comprising: synthesizing an FKBP-binding domain (FKBD)-containing precursor with a carboxylic acid terminal structure and an alkene terminal structure; assembling a tetrapeptide based on natural or unnatural amino acids using solid state peptide synthesis; bonding the tetrapeptide with the FKBD-containing precursor via its carboxylic acid terminal structure; and obtaining the macrocylic structure via a macrocyclization method.
 6. The method of claim 5, wherein the macrocyliczation method can be selected from the group consisting of ring-closing metathesis, macrolactamization, and click chemistry.
 7. A macrocyclic compound according to Formula (XII):

or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof wherein Ring A is a 5-10 membered aryl, cycloalkyl, heteroaryl or heterocycloalkyl, optionally substituted with 1-17 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin; J is independently at each occurrence selected from the group consisting of —C(O)NR⁶—.

R⁶ is each hydrogen, alkyl, arylalkyl,

R^(N) is aryl, alkyl, or arylalkyl; R′ is hydrogen, alkyl, arylalkyl, or haloalkyl; D is independently at each occurrence an oligonucleotide; L^(b) and L^(c) are independently at each occurrence selected from the group consisting of bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)OC(O)C₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)OC₁₋₆ alkylene (CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene (CH₂)n-, optionally substituted (CH₂)_(n)—S—C₁₋₆ alkylene (CH₂)_(n)—, and optionally substituted (CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or 2 groups independently selected from the group consisting of of halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; each R⁵ is independently hydrogen, alkyl, arylalkyl,

or and

R^(N) is aryl, alkyl, or arylalkyl; X is O, S or NR⁸, R⁸ is hydrogen, hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ are each independently hydrogen or alkyl; V¹ and V² are each independently

W is

Ring B is a 4-10 membered heterocycloalkyl, optionally substituted with 1-10 substituents, each of which is selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino, arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; R¹³ is hydrogen, hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is each independently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, arylalkyl, or heteroaryl; Z is bond,

R¹⁶ and R¹⁷ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸, CR¹⁸, N, or and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl; L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond, —O—, —NR¹⁹—, —SO—, —SO₂—, (CH₂)_(n)—,

or a linking group selected from Table 1; Ring C is a 5-6 membered heteroaryl, optionally substituted with 1-4 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxyl, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino and

wherein each R¹⁹, R²⁰, and R²¹ is independently is selected from the group consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴ are each independently hydrogen or alkyl; n is 0, 1, 2, 3, 4, 5 or
 6. 8. The tagged macrocyclic compound of claim 7, wherein the Effector Domain has Formula (XIIa):

each k^(a), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i) is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ is independently hydrogen, alkyl, arylalkyl or NR²⁵, wherein R²⁵ is hydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸ are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is independently selected from the group consisting of hydrogen, halo, amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl and


9. The tagged macrocyclic compound of claim 7, wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnatural amino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or
 10. 10. The tagged macrocyclic compound of claim 7, wherein the Effector Domain has Formula (XIIc):

wherein each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R²⁹ is a hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³² are each independently hydrogen or alkyl; X³ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-Q) alkynylene-, or

Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or
 2. 11. The tagged macrocyclic compound of claim 7, wherein the Effector Domain has Formula (XIId):

wherein X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or
 2. 12. The tagged macrocyclic compound of claim 7, wherein the Effector Domain has Formula (XIIe):

wherein R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or
 2. 13. The tagged macrocyclic compound of claim 7, wherein the Effector Domain has Formula (XIIf):

wherein X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; provided that when R is

L is ethylene, X is O, W is

V is

Z is

-L⁶-L⁷-L⁸-is

then -L¹-L²-L³-L⁴-L⁵-is not

and; wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring C substituted with at least one

or wherein at least one of the linking groups selected from Table 1 is substituted with at least one


14. A tagged compound of macrocyclic compound according to Formula (XII):

or a pharmaceutically acceptable salt, solvate, or stereoisomer thereof with a compound of Formula (XIV): Q′-L^(c)-D   Formula (XIV) wherein Ring A is a 5-10 membered aryl, cycloalkyl, heteroaryl or heterocycloalkyl, optionally substituted with 1-17 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino,

wherein

is a resin; L^(b) and L^(c) are independently selected from the group consisting of a bond, —O—, —S—, —OC(O)—, —C(O)O—, —(CH₂)_(n)C(O)—, —(CH₂)_(n)C(O)C(O)—, —(CH₂)_(n)NR⁵C(O)C(O)—, —NR⁵(CH₂)_(n)C(O)C(O)—, optionally substituted (CH₂)_(n)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)C(O)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)OC(O)C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)OC₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)NR⁵C₁₋₆ alkylene-(CH₂)_(n)—, optionally substituted (CH₂)_(n)—S—C₁₋₆ alkylene-(CH₂)_(n)—, and optionally substituted (CH₂CH₂O)_(n); wherein each alkylene is optionally substituted with 1 or 2 groups independently selected from the group consisting of of halo, hydroxy, haloalkyl, haloalkoxy, alkyl, alkoxy, amino, carboxyl, cyano, nitro, NHFmoc; wherein each R⁵ is independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; Q and Q′ are independently selected from the group consisting of —N₃, —C≡CH, NR⁶R⁷, —COOH, —ONH₂, —SH, —NH₂,

(C═O)R′,

wherein R⁶ and R⁷ is each independently hydrogen, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; and R′ is hydrogen, alkyl, arylalkyl, or haloalkyl; X is O, S or NR⁸, wherein R⁸ is hydrogen, hydroxy, OR⁹, NR¹⁰R¹¹, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R⁹, R¹⁰ and R¹¹ are each independently hydrogen or alkyl; V¹ and V² are each independently

W is

wherein Ring B is a 4-10 membered heterocycloalkyl, optionally substituted with 1-10 substituents, each of which is selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, cyano, haloalkyl, haloalkoxy, alkylthio, oxo, amino, alkylamino, dialkylamino, arylalkyl,

wherein R¹² is aryl, alkyl, or arylalkyl; wherein R¹³ is hydrogen, hydroxy, OR¹⁶, NR¹⁷R¹⁸, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; R¹⁴ and R¹⁵ is each independently hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, aryl, arylalkyl, or heteroaryl; Z is bond,

wherein R¹⁶ and R¹⁷ are each independently selected from the group consisting of of hydrogen, hydroxy, halo, alkyl, alkoxy, cycloalkyl, cyano, alkylthio, amino, alkylamino, and dialkylamino; K is O, CHR¹⁸, CR¹⁸, N, and NR¹⁸, wherein R¹⁸ is hydrogen or alkyl; L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ are each independently a bond, —O—, —NR¹⁹—, —SO—, —SO₂—, —(CH₂)_(n)—,

or a linking group selected from Table 1; wherein Ring C is a 5-6 membered heteroaryl, optionally substituted with 1-4 substituents, each of which is independently selected from the group consisting of hydrogen, hydroxy, halo, alkyl, alkoxy, haloalkyl, haloalkoxy, cyano, alkylthio, amino, alkylamino, dialkylamino and

wherein R¹⁹ is selected from the group consisting of hydrogen, hydroxy, OR²², NR²³R²⁴, alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²², R²³, and R²⁴ are each independently hydrogen or alkyl; n is 0, 1, 2, 3, 4, 5 or 6; wherein the Effector Domain has Formula (XIIa):

each k^(k), k^(b), k^(c), k^(d), k^(e), k^(f), k^(g), k^(h), and k^(i) is independently 0 or 1; each X^(a), X^(b), X^(c), X^(d), X^(e), X^(f), X^(g), X^(h), and X^(i) is independently a bond, —S—, —S—S—, —S(O)—, —S(O)₂—, substituted or unsubstituted —(C₁-C₃) alkylene-, —(C₂-C₄) alkenylene-, —(C₂-C₄) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; each R¹, R^(1a), R^(1b), R^(1c), R^(1d), R^(1e), R^(1f), R^(1g), R^(1h), R^(1i), and R⁴ is independently hydrogen, alkyl, arylalkyl or NR²⁵ wherein R²⁵ is hydrogen, hydroxy, OR²⁶, NR²⁷R²⁸, alkyl, arylalkyl, R

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R²⁶, R²⁷, and R²⁸ are each independently hydrogen or alkyl; each R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R^(2g), R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is independently selected from the group consisting of hydrogen, halo, amino, cyano, nitro, haloalkyl, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkylamino, optionally substituted dialkylamino, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, optionally substituted heteroarylalkyl, and

or wherein the Effector Domain has Formula (XIIb):

wherein each of AA¹, AA², . . . , and AA^(r) is an natural or unnatural amino acid residue; and r is 3, 4, 5, 6, 7, 8, 9, or 10; or wherein the Effector Domain has Formula (XIIc):

each t is independently an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; R²⁹ is hydrogen, hydroxy, OR³⁰, NR³¹R³², alkyl, arylalkyl,

wherein R^(N) is aryl, alkyl, or arylalkyl; wherein R³⁰, R³¹, and R³² are each independently hydrogen or alkyl; X³ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; or wherein the Effector Domain has Formula (XIId):

X⁴ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; or wherein the Effector Domain has Formula (XIIe):

R³³, R³⁴, R³⁵ and R³⁶ are each hydrogen or alkyl; X⁵ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; or wherein the Effector Domain has Formula (XIII):

X⁶ is substituted or unsubstituted —(C₁-C₆) alkylene-, —(C₂-C₆) alkenylene-, —(C₂-C₆) alkynylene-, or

wherein Ring E is phenyl or a 5-6 heteroaryl or heterocycloalkyl; wherein each w is independently 0, 1, or 2; and provided that when Ring A is

L^(a) is ethylene, X is O, W is

V is

V² is

Z is

-L⁶-L⁷-L⁸-is

and -L¹-L²-L³-L⁴-L⁵-is not

D is an oligonucleotide; wherein Ring A is substituted with at least one

or at least one of R², R³, R^(2a), R^(3a), R^(2b), R^(3b), R^(2c), R^(3c), R^(2d), R^(3d), R^(2e), R^(3e), R^(2f), R^(3f), R², R^(3g), R^(2h), R^(3h), R^(2i), and R^(3i) is

or at least one of L^(a), L¹, L², L³, L⁴, L⁵, L⁶, L⁷ and L⁸ is Ring C substituted with at least one

or wherein at least one of the linking groups selected from Table 1 is substituted with at least one 